Antiviral oligonucleotides having a conserved G4 core sequence

ABSTRACT

Modified oligonucleotides having a conserved G 4  sequence and a sufficient number of flanking nucleotides to significantly inhibit the activity of a virus or phospholipase A 2  or to modulate the telomere length of a chromosome are provided. G 4  quartet oligonucleotide structures are also provided. Methods of prophylaxis, diagnostics and therapeutics for viral-associated diseases and diseases associated with elevated levels of phospholipase A 2  are also provided. Methods of modulating telomere length of a chromosome are also provided; modulation of telomere length is believed to play a role in the aging process of a cell and in control of malignant cell growth.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.08/403,888 filed Jun. 12, 1995, which is the national phase of PCTApplication Serial No. PCT/US93/09297 filed Sep. 29, 1993, which is acontinuation-in-part of U.S. application Ser. No. 07/954,185 filed Sep.29, 1992, now abandoned, each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to the design and synthesis of oligonucleotideswhich can be used to inhibit the activity of viruses in vivo or in vitroand to treat viral-associated disease. These compounds can be usedeither prophylactically or therapeutically for diseases associated withviruses such as HIV, HSV, HCMV and influenza. Oligonucleotides capableof inhibiting phospholipase A₂ enzyme activity are also provided whichmay be useful for the treatment of inflammatory disorders, as well asneurological conditions. Oligonucleotides designed for the treatment ofcancer and to retard aging are also contemplated by this invention.

BACKGROUND OF THE INVENTION

Antivirals

There have been many approaches for inhibiting the activity of virusessuch as the human immunodeficiency virus (HIV), herpes simplex virus(HSV), human cytomegalovirus (HCMV) and influenza. Such prior artmethods include nucleoside analogs (e.g., HSV) and antisenseoligonucleotide therapies (e.g., HIV, influenza).

Prior attempts to inhibit HIV by various approaches have been made by anumber of researchers. For example, Zamecnik and coworkers have usedphosphodiester antisense oligonucleotides targeted to the reversetranscriptase primer site and to splice donor/acceptor sites, P. C.Zamecnik, J. Goodchild, Y. Taguchi, P. S. Sarin, Proc. Natl. Acad. Sci.USA 1986, 83, 4143. Goodchild and coworkers have made phosphodiesterantisense compounds targeted to the initiation sites for translation,the cap site, the polyadenylation signal, the 5′ repeat region, primerbinding site, splice sites and a site between the gag and pol genes. J.Goodchild, S. Agrawal, M. P. Civeira, P. S. Sarin, D. Sun, P. C.Zamecnik, Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 5507; U.S. Pat. No.4,806,463. Agrawal and coworkers have used chemically modified antisenseoligonucleotide analogs targeted to the cap and splice donor/acceptorsites. S. Agrawal, J. Goodchild, M. P. Civeira, A. H. Thornton, P. S.Sarin, P. C. Zamecnik, Proc. Nat'l. Acad. Sci. USA 1988, 85, 7079.Agrawal and coworkers have used antisense oligonucleotide analogstargeted to the splice donor/acceptor site inhibit HIV infection inearly infected and chronically infected cells. S. Agrawal, T. Ikeuchi,D. Sun, P. S. Sarin, A. Konopka, J. Maizel, Proc. Natl. Acad. Sci.U.S.A. 1989, 86, 7790.

Sarin and coworkers have also used chemically modified antisenseoligonucleotide analogs targeted to the HIV cap and splicedonor/acceptor sites. P. S. Sarin, S. Agrawal, M. P. Civeira, J.Goodchild, T. Ikeuchi, P. C. Zamecnik, Proc. Natl. Acad. Sci. U.S.A.1988, 85, 7448. Zaia and coworkers have also used an antisenseoligonucleotide analog targeted to a splice acceptor site to inhibitHIV. J. A. Zaia, J. J. Rossi, G. J. Murakawa, P. A. Spallone, D. A.Stephens, B. E. Kaplan, J. Virol. 1988, 62, 3914. Matsukura andcoworkers have synthesized antisense oligonucleotide analogs targeted tothe initiation of translation of the HIV rev gene mRNA. M. Matsukura, K.Shinozuka, G. Zon, Proc. Natl. Acad. Sci. USA 1987, 84, 7706; R. L.Letsinger, G. R. Zhang, D. K. Sun, T. Ikeuchi, P. S. Sarin, Proc. Natl.Acad. Sci. U.S.A. 1989, 86, 6553. Mori and coworkers have used adifferent antisense oligonucleotide analog targeted to the same regionas Matsukura. K. Mori, C. Boiziau, C. Cazenave, Nucleic Acids Res. 1989,17, 8207. Shibahara and coworkers have used antisense oligonucleotideanalogs targeted to a splice acceptor site as well as the reversetranscriptase primer binding site. S. Shibahara, S. Mukai, H. Morisawa,H. Nakashima, S. Kobayashi, N. Yamamoto, Nucl. Acids Res. 1989, 17, 239.Letsinger and coworkers have synthesized and tested a oligonucleotideanalogs with conjugated cholesterol targeted to a splice site. K. Mori,C. Boiziau, C. Cazenave, Nucleic Acids Res. 1989, 17, 8207. Stevensonand Iversen have conjugated polylysine to antisense oligonucleotideanalogs targeted to the splice donor and the 5′-end of the first exon ofthe HIV tat gene. M. Stevenson, P. L. Iversen, J. Gen. Virol. 1989, 70,2673. Buck and coworkers have described the use of phosphate-methylatedDNA oligonucleotides targeted to HIV mRNA and DNA. H. M. Buck, L. H.Koole, M. H. P. van Gendersen, L. Smith, J. L. M. C. Green, S. Jurriaansand J. Goudsmit, Science 1990, 248, 208-212.

These prior attempts at inhibiting HIV activity have largely focused onthe nature of the chemical modification used in the oligonucleotideanalog. Although each of the above publications have reported somedegree of success in inhibiting some function of the virus, a generaltherapeutic scheme to target HIV and other viruses has not been found.Accordingly, there has been and continues to be a long-felt need for thedesign of compositions which are capable of effective, therapeutic use.

Currently, nucleoside analogs are the preferred therapeutic agents forherpes (HSV) infections. A number of pyrimidine deoxyribonucleosidecompounds have a specific affinity for the virus-encoded thymidine(dCyd) kinase enzyme. The specificity of action of these compoundsconfines the phosphorylation and antiviral activity of these compoundsto virus-infected cells. A number of drugs from this class, e.g.,5-iodo-dUrd (IDU), 5-trifluoro-methyl-dUrd (FMAU), 5-ethyl-dUrd (EDU),(E)-5-(2-bromovinyl)-dUrd (BVDU), 5-iodo-dCyd (IDC), and5-trifluoromethyl-dUrd (TFT), are either in clinical use or likely tobecome available for clinical use in the near future. IDU is amoderately effective topical antiviral agent when applied to HSVgingivostomatitis and ocular stromal keratitis; however, its use incontrolled clinical studies of HSV encephalitis revealed a high toxicityassociated with IDU treatment. Although the antiviral specificity of5-arabinofuranosyl cytosine (Ara-C) was initially promising, itsclinical history has paralleled that of IDU. The clinical appearance ofHSV strains which are deficient in their ability to synthesize the viralthymidine kinase has generated further concern over the future efficacyof this class of compounds.

The utility of a number of viral targets has been defined for anti-HSVcompound development. Studies with thiosemicarbazone compounds havedemonstrated that inhibition of the viral ribonucleotide reductaseenzyme is an effective means of inhibiting replication of HSV in vitro.Further, a number of purine nucleosides which interfere with viral DNAreplication have been approved for treatment of human HSV infections.9-(β-D-arabinofuranosyl)adenine (Ara-A) has been used for treatment ofHSV-1 keratitis, HSV-1 encephalitis and neonatal herpes infections.Reports of clinical efficacy are contradictory and a major disadvantagefor practical use is the extremely poor solubility of Ara-A in water.9-(2-hydroxyethoxymethyl)guanine (Acyclovir, ACV) is of major interest.In humans, ACV has been used successfully in the therapy of localizedand disseminated HSV infections. However there appear to be both theexistence of drug-resistant viral mutants and negative results indouble-blind studies of HSV-1 treatment with ACV. ACV, like Ara-A, ispoorly soluble in water (0.2%) and this physical characteristic limitsthe application forms for ACV. The practical application of purinenucleoside analogs in an extended clinical situation suffers from theirinherently efficient catabolism, which not only lowers the biologicalactivity of the drug but also may result in the formation of toxiccatabolites.

The effective anti-HSV compounds currently in use or clinical testingare nucleoside analogs. The efficacy of these compounds is diminished bytheir inherently poor solubility in aqueous solutions, rapidintracellular catabolism and high cellular toxicities. An additionalcaveat to the long-term use of any given nucleoside analogue is therecent detection of clinical isolates of HSV which are resistant toinhibition by nucleoside compounds which were being administered inclinical trials. Antiviral oligonucleotides offer the potential ofbetter compound solubilities, lower cellular toxicities and lesssensitivity to nucleotide point mutations in the target gene than thosetypical of the nucleoside analogs.

Effective therapy for cytomegalovirus (CMV) has not yet been developeddespite studies on a number of antivirals. Interferon, transfer factor,adenine arabinoside (Ara-A), acycloguanosine (Acyclovir, ACV) andcertain combinations of these drugs have been ineffective in controllingCMV infection. Based on preclinical and clinical data, foscarnet (PFA)and ganciclovir (DHPG) show limited potential as antiviral agents. PFAtreatment has resulted in the resolution of CMV retinitis in five AIDSpatients. DHPG studies have shown efficacy against CMV retinitis orcolitis. DHPG seems to be well tolerated by treated individuals, but theappearance of a reversible neutropenia, the emergence of resistantstrains of CMV upon long-term administration, and the lack of efficacyagainst CMV pneumonitis limit the long term applications of thiscompound. The development of more effective and less-toxic therapeuticcompounds and methods is needed for both acute and chronic use.

Classical therapeutics has generally focused upon interactions withproteins in efforts to moderate their disease-causing ordisease-potentiating functions. Such therapeutic approaches have failedfor cytomegalovirus infections. Therefore, there is an unmet need foreffective compositions capable of inhibiting cytomegalovirus activity.

There are several drugs available which have some activity against theinfluenza virus prophylactically. None, however, are effective againstinfluenza type B. Moreover, they are generally of very limited usetherapeutically and have not been widely used in treating the diseaseafter the onset of symptoms. Accordingly, there is a world-wide need forimproved therapeutic agents for the treatment of influenza virusinfections.

Prior attempts at the inhibition of influenza virus using antisenseoligonucleotides have been reported. Leiter and co-workers have targetedphosphodiester and phosphorothioate oligonucleotides to influenza A andinfluenza C viruses. Leiter, J., Agrawal, S., Palese, P. & Zamecnik, P.C., Proc. Natl. Acad. Sci. USA; 1990, 87, 3430-3434. These workerstargeted the polymerase PB1 gene and mRNA in the vRNA 3′ region and mRNA5′ region, respectively. Sequence-specific inhibition of influenza A wasnot observed although some specific inhibition of influenza C was noted.

Zerial and co-workers have reported inhibition of influenza A virus byoligonucleotides coincidentally linked to an intercalating agent.Zerial, A., Thuong, N. T. & Helene, C., Nucleic Acids Res. 1987, 57,9909-9919. Zerial et al. targeted the 3′ terminal sequence of 8 vRNAsegments. Their oligonucleotide analog was reported to inhibit thecytopathic effects of the virus in cell culture.

Kabanov and co-workers have synthesized an oligonucleotide complementaryto the loop-forming site of RNA encoding RNA polymerase 3. Kabanov, A.V., Vinogradov, S. V., Ovcharenko, A. V., Krivonos, A. V.,Melik-Nubarov, N. S., Kiselev, V. I., Severin, E. S., FEB; 1990, 259,327-330. Their oligonucleotide was conjugated to a undecyl residue atthe 5′ terminal phosphate group. They found that their oligonucleotideinhibited influenza A virus infection in MDCK cells.

Although each of the foregoing workers reported some degree of successin inhibiting some function of an influenza virus, a general therapeuticscheme to target influenza viruses has not been found. Moreover,improved efficacy is required in influenza virus therapeutics.Accordingly, there has been and continues to be a long-felt need for thedesign of oligonucleotides which are capable of effective therapeuticuse.

Phospholipase A₂ Enzyme Activity

Phospholipase A₂ is a family of lipolytic enzymes which hydrolyzemembrane phospholipids. Phospholipase A₂ catalyzes the hydrolysis of thesn-2 bond of phospholipids resulting in the production of free fattyacid and lysophospholipids. Several types of phospholipase A₂ enzymeshave been cloned and sequenced from human cells. However, there isbiochemical evidence that additional forms of phospholipase A₂ exists.Mammalian secreted phospholipase A₂ shares strong sequence similaritieswith phospholipase A₂ isolated from the venom of poisonous snakes.Secreted forms of phospholipase A₂ have been grouped into two categoriesbased upon the position of cysteine residues in the protein. Type Iphospholipase A₂ includes enzymes isolated from the venoms of Elapidae(cobras), Hydrophidae (sea snakes) and the mammalian pancreatic enzyme.Type II phospholipase A₂ includes enzymes isolated from the venoms ofCrotalidae (rattlesnakes and pit vipers), Viperidae (old world vipers)and an enzyme secreted from platelets and other mammalian cells.

Much interest has been generated in mammalian type II phospholipase A₂,in that elevated concentrations of the enzyme have been detected in avariety of inflammatory disorders including rheumatoid arthritis,inflammatory bowel disease, and septic shock as well as neurologicalconditions such as schizophrenia, Pruzanski, W., Keystone, E. C.,Stemby, B., Bombardier, C., Snow, K. M., and Vadas, P. J. Rheumatol.1988, 15, 1351; Pruzanski and Vadas J. Rheumatol. 1988, 15, 11; Oliason,G., Sjodahl, R., and Tagesson, C. Digestion 1988, 41, 136; Vadas et al.Crit. Care Med. 1988, 16, 1; Gattaz, W. F., Hubner, C. v.K., Nevalainen,T. J., Thuren, T., and Kinnunen, P. K. J. Biol. Psychiatry 1990, 28,495. It has been recently demonstrated that secretion of type IIphospholipase A₂ is induced by a variety of proinflammatory cytokinessuch as interleukin-1, interleukin 6, tumor necrosis factor,interferon-γ, and bacterial lipopolysaccharide. Hulkower, K., Hope, W.C., Chen, T., Anderson, C. M., Coffey, J. W., and Morgan, D. W.,Biochem. Biophys. Res. Comm. 1992, 184, 712; Crowl, R. M., Stoller, T.J., Conroy, R. R. and Stoner, C. R., J. Biol. Chem. 1991, 266, 2647;Schalkwijk, C., Pfeilschafter, J., Marki, F., and van den Bosch, J.,Biochem. Biophys. Res. Comm. 1991, 174, 268; Gilman, S. C. and Chang,J., J. Rheumatol. 1990, 17, 1392; Oka, S. and Arita, H., J. Biol. Chem.1991, 266, 9956. Anti-inflammatory agents such as transforming growthfactor-β and glucocorticoids have been found to inhibit secretion oftype II phospholipase A₂. Oka, S. and Arita, H., J. Biol. Chem. 1991,266, 9956; Schalkwijk, C., Pfeilschifter, J., Marki, F. and van denBosch, H., J. Biol. Chem. 1992, 267, 8846. Type II phospholipase A₂ hasbeen demonstrated to be secreted from a variety of cell types includingplatelets, chrondrocytes, synoviocytes, vascular smooth muscle cells,renal mesangial cells, and keratinocytes. Kramer, R. M., Hession, C.,Johansen, B., Hayes, G., McGray, P., Chow, E. P., Tizard, R. andPepinsky, R. B., J. Biol. Chem. 1989, 264, 5768; Gilman, S. C. andChang, J., J. Rheumatol. 1990, 17, 1392; Gilman, S. C., Chang, J.,Zeigler, P. R., Uhl, J. and Mochan, E., Arthritis and Rheumatol. 1988,31, 126; Nakano, T., Ohara, O., Teraoka, H. and Arita, H., FEBS Lett.,1990, 261, 171; Schalkwijk, C., Pfeilschifter, J., Marki, F. and van denBosch, H. Biochem. Biophys. Res. Comm. 1991, 174, 268.

A role of type II phospholipase A₂ in promoting some of thepathophysiology observed in chronic inflammatory disorders was suggestedbecause direct injection of type II phospholipase A₂ produced profoundinflammatory reactions when injected intradermally or in the articularspace in rabbits, Pruzanski, W., Vadas, P., Fomasier, V., J. Invest.Dermatol. 1986, 86, 380-383; Bomalaski, J. S., Lawton, P., and Browning,J. L., J. Immunol. 1991, 146, 3904; Vadas, P., Pruanski, W., Kim, J. andFormasier, V., Am. J. Pathol. 1989, 134, 807. Denaturation of theprotein prior to injection was found to block the proinflammatoryactivity.

Because of these findings, there is interest in identifying potent andselective inhibitors of type II phospholipase A₂. To date, efforts atidentifying non toxic and selective inhibitors of type II phospholipaseA₂ have met with little success. Therefore, there is an unmet need toidentify effective inhibitors of phospholipase A₂ activity.

Modulation of Telomere Length

It has been recognized that telomeres, long chains of repeatednucleotides located at the tip of each chromosome, play a role in theprotection and organization of the chromosome. In human cells, thesequence TTAGGG is repeated hundreds to thousands of times at both endsof every chromosome, depending on cell type and age. Harley, C. B. etal., Nature, 1990, 345, 458-460; Hastie, N. D. et al., Nature, 1990,346, 866-868. Telomeres also appear to have a role in cell aging whichhas broad implications for the study of aging and cell immortality thatis manifested by cancerous cells.

Researchers have determined that telomere length is reduced whenever acell divides and it has been suggested that telomere length controls thenumber of divisions before a cell's innate lifespan is spent. Harley, C.B. et al., Nature, 1990, 345, 458-460; Hastie, N. D. et al., Nature,1990, 346, 866-868. For example, normal human cells divide between70-100 times and appear to lose about 50 nucleotides of their telomereswith each division. Some researchers have suggested that there is astrong correlation between telomere length and the aging of the entirehuman being. Greider, C. W., Curr. Opinion Cell Biol., 1991, 3, 444-451.Other studies have shown that telomeres undergo a dramatictransformation during the genesis and progression of cancer. Hastie, N.D. et al., Nature 1990, 346, 866-868. For example, it has been reportedthat when a cell becomes malignant, the telomeres become shortened witheach cell division. Hastie, N. D. et al., Nature 1990, 346, 866-868.Experiments by Greider and Bacchetti and their colleagues have shownthat at a very advanced and aggressive stage of tumor development,telomere shrinking may cease or even reverse. Counter, C. M. et al.,EMBO J. 1992, 11, 1921-1929. It has been suggested, therefore, thattelomere blockers may be useful for cancer therapy. In vitro studieshave also shown that telomere length can be altered by electroporationof linearized vector containing human chromosome fragments into hybridhuman-hamster cell lines. Chromosome fragments consisted ofapproximately 500 base pairs of the human telomeric repeat sequenceTTAGGG and related variants such as TTGGGG, along with adjacent GC-richrepetitive sequences. Farr, C. et al., Proc. Natl. Acad. Sci. USA 1992,88, 7006-7010. While this research suggests that telomere length affectscell division, no effective method for control of the aging process orcancer has been discovered. Therefore, there is an unmet need toidentify effective modulators of telomere length.

Guanosine nucleotides, both as mononucleotides and in oligonucleotidesor polynucleotides, are able to form arrays known as guanine quartets orG-quartets. For review, see Williamson, J. R., (1993) Curr. Opin.Struct. Biol. 3:357-362. G-quartets have been known for years, althoughinterest has increased in the past several years because of theirpossible role in telomere structure and function. One analyticalapproach to this area is the study of structures formed by shortoligonucleotides containing clusters of guanosines, such as GGGGTTTTGGGG(SEQ ID NO:143), GGGTTTTGGG (SEQ ID NO:144), UGGGGU, GGGGGTTTTT (SEQ IDNO:145), TTAGGG, TTGGGG and others reviewed by Williamson; TTGGGGTTdescribed by Shida et al. (Shida, T., Yokoyama, K., Tamai, S., and J.Sekiguchi (1991) Chem. Pharm. Bull. 39:2207-2211), and others.

It has now been discovered that in addition to their natural role (intelomeres, for example, though there may be others), oligonucleotideswhich form G-quartets and oligonucleotides containing clusters of G'sare useful for inhibiting viral gene expression and viral growth and forinhibiting PLA₂ enzyme activity, and may also be useful as modulators oftelomere length. Chemical modification of the oligonucleotides for suchuse is desirable and, in some cases, necessary for maximum activity.

Oligonucleotides containing only G and T have been designed to formtriple strands with purine-rich promotor elements to inhibittranscription. These triplex-forming oligonucleotides (TFOs), 28 to 54nucleotides in length, have been used to inhibit expression of theoncogene c-erb B2/neu (WO 93/09788, Hogan). Amine-modified TFOs 31-38nucleotides long have also been used to inhibit transcription of HIV.McShan, W. M. et al. (1992) J. Biol. Chem. 267:5712-5721.

OBJECTS OF THE INVENTION

It is an object of the invention to provide oligonucleotides capable ofinhibiting the activity of a virus.

It is another object of the invention to provide methods of prophylaxis,diagnostics and therapeutics for viral-associated diseases such as HIV,HSV, HCMV and influenza.

It is a further object of the invention to provide oligonucleotidescapable of inhibiting phospholipase A₂.

Yet another object of the invention is to provide methods ofprophylaxis, diagnostics and therapeutics for the treatment ofinflammatory disorders, as well as neurological conditions associatedwith elevated levels of phospholipase A₂.

It is another object of the invention to provide oligonucleotides formodulating telomere length on chromosomes.

It is another object of the invention to provide oligonucleotidecomplexes capable of inhibiting HIV.

These and other objects will become apparent to persons of ordinaryskill in the art from a review of the instant specification and appendedclaims.

SUMMARY OF THE INVENTION

It has been discovered that oligonucleotides containing the sequenceGGGG (G₄), denominated herein as a conserved G₄ core sequence, haveantiviral activity against a number of viruses including but not limitedto HIV, HSV, HCMV, and influenza virus. A sequence containing 4 guanines(G's) or 2 stretches of 3 G's has been found to be effective forsignificant antiviral activity. It has also been discovered thatoligonucleotides containing a conserved G₄ core sequence or twostretches of 3 G's are effective inhibitors of phospholipase A₂activity. It is also believed that such oligonucleotides could be usefulfor modulation of telomere length on chromosomes.

The formula for an active sequence is generally (N_(X)G₄N_(Y))_(Q) or(G₃₋₄N_(X)G₃₋₄)_(Q) wherein X and Y are 1-8, and Q is 1-4. The sequence(N_(X)G₃₋₄)_(Q)N_(X) wherein X is 1-8 and Q is 1-6 has also been foundto be useful in some embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing anti-HSV activity of G₄ oligonucleotides asmeasured by virus yield assay. Cells were treated with oligonucleotideat dose of 3 μM or 10 μM. Viral titers are shown as a percentage ofvirus titer from untreated, infected cells. All oligonucleotides testedcontain a phosphorothioate backbone except for those noted with a P═O.

FIG. 2 is a graph showing dose-dependent anti-HSV activity of G₄oligonucleotides 5651 (SEQ ID NO: 35), 5652 (SEQ ID NO: 37), 5653 (SEQID NO: 38), 5676 (SEQ ID NO: 39), and 4015 (SEQ ID NO: 21). 3383 (SEQ IDNO: 122) is a negative control oligonucleotide. ACV is Acyclovir(positive control).

FIG. 3 is a graph showing anti-influenza activity of G₄ oligonucleotidesas measured by virus yield assay. Oligonucleotides were tested at asingle dose of 10 mM. Virus titer is expressed as a percentage of thetiter obtained from untreated, infected cells.

FIG. 4 is a graph showing the inhibition of phospholipase A₂ by various2′-substituted oligonucleotides.

FIG. 5 is a graph showing the effect of ISIS 3196 (SEQ ID NO: 47) onenzyme activity of phospholipase A₂ isolated from different sources.

FIG. 6 is a graph showing the results of an experiment wherein humanphospholipase A₂ was incubated with increasing amounts of E. colisubstrate in the presence of oligonucleotides ISIS 3196 (SEQ ID NO: 47)and ISIS 3481 (SEQ ID NO: 77).

FIG. 7 is a line graph showing the effect of time of oligonucleotideaddition on HSV-1 inhibition.

FIG. 8 is a line graph showing activity of ISIS 4015 and 2′-O-propylgapped phosphorothioate oligonucleotides against HSV-1.

FIG. 9 is a line graph showing activity of ISIS 3657 and 2′-O-propylphosphorothioate oligonucleotides against HSV-1.

FIG. 10 is a three-dimensional bar graph showing effects on HSV-1 ofISIS 4015 and TFT separately and in combination.

FIG. 11 is a three-dimensional bar graph showing effects on HSV-1 ofISIS 4015 and ACV separately and in combination.

FIG. 12 is a line graph showing antiviral activity of G-stringoligonucleotides 5684, 5058, 5060, 6170 and 4015.

FIG. 13 is a line plot showing dissociation of ISIS 5320 tetramermonitored by size exclusion chromatography over a period of 1 to 131days.

FIG. 14 is an autoradiogram of a gel electrophoresis experiment showinga pattern characteristic of a parallel-stranded tetramer. Lane 1: ISIS5320 (T₂G₄T₂) alone. Lane 2: ISIS 5320 incubated with T₁₃G₄G₄ (SEQ IDNO:146). Lane 3. T₁₃G₄T₄ (SEQ ID NO:142) alone.

FIG. 15 is a line graph showing dissociation of tetramers formed byphosphorothioate ISIS 5320 in Na+ (squares), ISIS 5320 in K+ (diamonds)and the phosphodiester version (circles) over a period of days.

FIG. 16 is a line graph showing binding of ISIS 5320 to gp120, measuredby absorbance at 405 nm.

FIG. 17 is a line graph showing that dextran sulfate is a competitiveinhibitor of binding of biotinylated ISIS 5320 to gp120.

FIG. 18 is a line graph showing that ISIS 5320 blocks binding of anantibody specific for the V3 loop of gp120 (solid line) but notantibodies specific for CD44 (even dashes) or CD4 (uneven dashes), asdetermined by immunofluorescent flow cytometry.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It has been discovered that oligonucleotides containing the sequenceGGGG (G₄,) where G is a guanine-containing nucleotide or analog, anddenominated herein as a conserved G₄ sequence, have potent antiviralactivity and can be effective inhibitors of phospholipase A₂ activityand modulators of telomere length on chromosomes. In the context of thisinvention, the term “oligonucleotide” refers to an oligomer or polymerof ribonucleic acid or deoxyribonucleic acid. This term includesoligomers consisting of naturally occurring bases, sugars and intersugar(backbone) linkages as well as oligomers having non-naturally occurringportions which function similarly. Such chemically modified orsubstituted oligonucleotides are often preferred over native formsbecause of properties such as, for example, enhanced cellular uptake andincreased stability in the presence of nucleases.

Specific examples of some preferred oligonucleotides envisioned for thisinvention may contain modified intersugar linkages (backbones) such asphosphorothioates, phosphotriesters, methyl phosphonates, chain alkyl orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are those withCH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂and O—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—P—O—CH₂). Alsopreferred are oligonucleotides having morpholino backbone structures.Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506. In otherpreferred embodiments, such as the protein-nucleic acid (PNA) backbone,the phosphodiester backbone of the oligonucleotide may be replaced witha polyamide backbone, the bases being bound directly or indirectly tothe aza nitrogen atoms of the polyamide backbone. P. E. Nielsen, M.Egholm, R. H. Berg, O. Buchardt, Science 1991, 254, 1497. Otherpreferred oligonucleotides may contain modified sugar moietiescomprising one of the following at the 2′ position: OH, SH, SCH₃, F,OCN, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ toC₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br;CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; fluorescein; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A fluorescein moietymay be added to the 5′ end of the oligonucleotide. Oligonucleotides mayalso have sugar mimetics such as cyclobutyls in place of thepentofuranosyl group. Alpha (α) anomers instead of the standard beta (β)nucleotides may also be used. Modified bases such as 7-deaza-7-methylguanosine may be used. A “universal” base such as inosine may also besubstituted for A, C, G, T or U.

Chimeric oligonucleotides can also be employed; these molecules containtwo or more chemically distinct regions, each comprising at least onenucleotide. These oligonucleotides typically contain a region ofmodified nucleotides that confer one or more beneficial properties (suchas, for example, increased nuclease resistance, increased uptake intocells, increased binding affinity for the target molecule) and anunmodified region that retains the ability to direct RNase H cleavage.

The oligonucleotides in accordance with this invention preferablycomprise from about 6 to about 27 nucleic acid base units. It ispreferred that such oligonucleotides have from about 6 to 24 nucleicacid base units. As will be appreciated, a nucleic acid base unit is abase-sugar combination suitably bound to adjacent nucleic acid base unitthrough phosphodiester or other bonds.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed, however the actual synthesis of theoligonucleotides are well within the talents of the routineer. It isalso well known to use similar techniques to prepare otheroligonucleotides such as the phosphorothioates and alkylatedderivatives.

Compounds with more than four G's in a row are active, but four in a rowor two or more runs of three G's in a row have been found to be requiredfor significant inhibitory activity. In the context of this invention, asignificant level of inhibitory activity means at least 50% inhibitionof activity as measured in an appropriate, standard assay. Such assaysare well known to those skilled in the art. Although the conserved G₄core sequence or G₄ pharmacophore is necessary, sequences flanking theG₄ core sequence have been found to play an important role in inhibitoryactivity because it has been found that activity can be modulated bysubstituting or deleting the surrounding sequences. In the context ofthis invention, the term “modulate” means increased or decreased.

The essential feature of the invention is a conserved G₄ core sequenceand a sufficient number of additional flanking bases to significantlyinhibit activity. It has also been discovered that analogs are toleratedin the backbone. For example, deoxy, phosphorothioate and 2′-O-Methylanalogs have been evaluated.

The formula for an active sequence is:(N_(x)G₄N_(y))_(Q) or (G₄N_(x)G₄)_(Q)where G=a guanine-containing nucleotide or analog, N=any nucleotide,X=1-8, Y=1-8, and Q=1-4. In some embodiments of the present invention,the sequence (N_(X)G₃₋₄)_(Q)N_(X) wherein X is 1-8 and Q is 1-6 has beenfound to be active.Antivirals

A series of oligonucleotides containing G₄ or 2 stretches of G₃ weretested for inhibition of HSV replication. Antiviral activity wasdetermined by ELISA. The results are shown in Table 1. Activity is shownas E.C.₅₀, which is the concentration of oligonucleotide which provides50% inhibition of HSV replication relative to control infected cells.Oligonucleotides were generally tested at doses of 3 μM and lower. TABLE1 Oligonucleotide inhibition of HSV replication SEQ ISIS EC50 ID NOSEQUENCE LENGTH COMPOSITION (μm) NO 1220 CAC GAA AGG CAT 21 MER P = S0.24, 1 GAC CGG GGC 0.16 4881 GAA AGG CAT GAC 18 MER P = S 0.7, 2 CGGGGC 0.65 4874 AGG CAT GAC CGG 15 MER P = S 1.1, 3 GGC 0.83 4873 CAT GACCGG GGC 12 MER P = S 1.4, 4 1.0 5305 CAC GAA AGG CAT 19 MER P = S >3.0 5GAC CGG G 5301 CAC GAA AGG CAT 18 MER P = S >3.0 6 GAC CGG 5302 CAC GAAAGG CAT 15 MER P = S >3.0 7 GAC 4274 CAT GGC GGG ACT 21 MER P = S 0.15,8 ACG GGG GCC 0.15 4882 CAT GGC GGG ACT 15 MER P = S 1.7, 9 ACG 1.4 4851T GGC GGG ACT ACG 18 MER P = S 0.55, 10 GGG GC 0.5 4872 GGC GGG ACT ACG15 MER P = S 1.9, 11 GGG 1.7 4338 ACC GCC AGG GGA 21 MER P = S 0.2, 12ATC CGT CAT 0.2 4883 GCC AGG GGA ATC 18 MER P = S 1.8, 13 CGT CAT 1.84889 AGG GGA ATC CGT 15 MER P = S 2.0, 14 CAT 2.0 4890 GCC AGG GGA ATC15 MER P = S 0.75, 15 CGT 0.7 3657 CAT CGC CGA TGC 21 MER P = S 0.2 16GGG GCG ATC 4891 CAT CGC CGA TGC 18 MER P = S 0.3 17 GGG GCG 4894 CATCGC CGA TCG 15 MER P = S >3.0 18 GGG 4895 CGC CGA TGC GGG 15 MER P = S0.55 19 GCG 4896 GC CGA TGC GGG G 12 MER P = S 1.2 20 4015 GTT GGA GACCGG 21 MER P = S 0.22, 21 GGT TGG GG 0.22 4549 GGA GAC CGG GGT 17 MER P= S 0.22, 22 TGG GG 0.27 5365 GA GAC CGG GGT TGG 16 MER P = S 0.47 23 GG4885 A GAC CGG GGT TGG 15 MER P = S 0.42, 24 GG 0.51 5356 CGG GGT TGG GG11 MER P = S 0.7 25 4717 GG GGT TGG GG 10 MER P = S 0.6 26 5544 TGG GG 5MER P = S >3.0 4803 GG GG 4 MER P = S >3.0 4771 GTT GGA GAC CGG 17 MER P= S 0.7 27 GGT TG 4398 CAC GGG GTC GCC 20 MER P = S 0.1 28 GAT GAA CC4772 GGG GTC GCC GAT 17 MER P = S 0.4 29 GAA CC 4773 CAC GGG GTC GCC 17MER P = S 0.2 30 GAT GA 4897 CAC GGG GTC GCC 15 MER P = S 0.13 31 GAT4721 CAC GGG GTC G 10 MER P = S 0.4 32 5366 TTG GGG TTG GGG 25 MER P = S0.16 33 TTG GGG TTG GGGG 5367 TTG GGG TTG GGG 25 MER P = O >4.0 34 TTGGGG TTG GGGG 5651 TT GGGG TT GGGG TT 24 MER P = S 0.17 35 GGGG TT GGGG5677 GGGG TT GGGG TT 22 MER P = S 0.2 36 GGGG TT GGGG 5652 TT GGGG TTGGGG TT 20 MER P = S 0.16 37 GGGG TT 5653 TT GGGG TT GGGG TT 18 MER P =S 0.2 38 GGGG 5676 GGGG TT GGGG TT 16 MER P = S 0.23 39 GGGG 5675 TTGGGG TT GGGG TT 14 MER P = S 0.42 40 5674 TT GGGG TT GGGG 12 MER P = S1.5 41 5320 TT GGGG TT 8 MER P = S >3.0 5739 TT GGGG 6 MER P = S >3.05544 T GGGG 5 MER P = S >3.0 4803 GGGG 4 MER P = S >3.0 4560 GGGG C GGGGC GGGG 21 MER P = S 0.18 42 C GGGG C G 5649 TT GGGG TT GGGG TT 24 MER P= O >3.0 43 GGGG TT GGGG 5670 GGGG TT GGGG TT 22 MER P = O >3.0 44 GGGGTT GGGG 5650 TT GGGG TT GGGG TT 20 MER P = O >3.0 45 GGGG TT 5590 GGGGTT GGGG 10 MER P = O >3.0 46 3196 GGG T GGG T ATA G 21 MER P = S 0.2 47AAG G GCT CC 4664 GGG T GGG T ATA G 18 MER P = S 0.2 48 AAG G GC 4671GGG T GGG T ATA 15 MER P = S 0.4 49 GAA G 4672 GGG T GGG T ATA G 12 MERP = S 0.2 50 4692 T GGG T ATA G AAG 18 MER P = S 1.5 51 GGC TCC 4693 G TATA G AAG GGC 15 MER P = S >3.0 52 TCC 4694 TA G AAG GGC TCC 12 MER P =S >3.0 53 5753 UUG GGG UU 8 MER O-Me >3.0 5756 TTA GGG TT 8 MER P =S >3.0 5755 CCC CGG GG 8 MER P = S >3.0

Oligonucleotides containing G₄ sequences were also tested for antiviralactivity against human cytomegalovirus (HCMV, Table 2) and influenzavirus (FIG. 3). Again, antiviral activity was determined by ELISA andI.C.₅₀'s shown are expressed as a percent of virus titer from untreatedcontrols. TABLE 2 Antiviral Activity of Oligonucleotides Tested AgainstHCMV SEQ ISIS I.C.₅₀ ID NO SEQUENCE COMP. (μm) NO 4015 GTT GGA GAC CGGGGT TGG GG P = S 0.17 21 4717 GGG GTT GGG G P = S 1.0 26 5366 TTG GGGTTG GGG TTG GGG TTG P = S 0.1 33 GGG G 4560 GGG GCG GGG CGG GGC GGG GCGP = S 0.15 42 5367 TTG GGG TTG GGG TTG GGG TTG P = O >2.0 34 GGG G

In the experiments it was found that the G₄ core was necessary forantiviral activity. Nucleotides surrounding G₄ contributed to antiviralactivity since deletion of nucleotides flanking the G₄ core decreasedantiviral activity. Oligonucleotides containing phosphorothioatebackbones were most active against HSV in these experiments. Compoundscontaining a phosphodiester backbone were found to be generally inactivein these studies. Compounds with various multiples of G₄ and T₂demonstrated comparable activity against HSV. However, T₂G₄T₂G₄ was lessactive and T₂G₄T₂ was inactive. It is believed that it is not necessarythat G₄ be flanked by T₂ since a compound containing multiples of G₄Chad antiviral activity similar to that observed for G₄T₂.Oligonucleotides containing G₄ also showed antiviral activity in a HSVvirus yield assay, as shown in FIG. 1. T₂G₄T₂G₄T₂G₄T₂G₄ (ISIS #5651, SEQID NO: 35) showed greater antiviral activity than did Acyclovir at adose of 3 mM. Several G₄ oligonucleotides were subsequently shown toreduce virus yield in a dose-dependent manner (FIG. 2). Oligonucleotidescontaining G₄ also showed significant antiviral activity against HCMV(Table 2) and influenza virus (FIG. 3). Control compounds without G₄sequences did not show antiviral activity.

A series of compounds comprising G₄ were tested for HIV activity. Theresults are shown in Table 3. TABLE 3 Oligonucleotide inhibition of HIVTI SEQ ISIS IC50 TC50 (TC50/ ID NO SEQUENCE COMPOSITION (μM) (μM) IC50)NO 5274 GCC CCC TA P = O INACTIVE 5273 GCT TTT TA P = O INACTIVE 5272GCG GGG TA P = O INACTIVE 5271 GCA AAA TA P = O INACTIVE 5312 GCG GGG TAP = S 1.3 5311 GCA AAA TA P = S INACTIVE >200 5307 GCT TTT TA P = SINACTIVE 5306 GCC CCC TA P = S INACTIVE 5319 TCG GGG TT P = S 1 5059 GGGGGG TA P = S 0.53 5325 CGG GGG TA P = S 1.1 5321 CCG GGG CC P = S 1.75753 UUG GGG UU O-ME, INACTIVE >>50 P = O 5058 GC GGGG TA P = S, 1.5 >255756 TTA GGG TT P = S 29 >50 5755 CCC CGG GG P = S 34 >>50 5543 TTT GGGTT P = S INACTIVE 5542 TTT GG TTT P = S INACTIVE 5544 TGGGG P = S 5 4560GGG GCG GGG CGG GGC P = S 0.14 42 GGG GCG 4721 CAC GGG GTC G P = S 0.21,142 546 32 0.26 4338 ACC GCC AGG GGA ATC P = S 0.42 12 CGT CAT 4897 CACGGG GTC GCC GAT P = S 0.43 31 3657 CAT CGC CGA TGC GGG P = S 0.43 16 GCGATC 4873 CAT GAC CGG GGC P = S 1 4 5366 TTG GGG TTG GGG TTG P = S 0.08,22 220 33 GGG TTG GGGG 0.1 5651 TT GGGG TT GGGG TT P = S 0.1, 19, 175 35GGGG TT GGGG .18 19 5677 GGGG TT GGGG TT P = S 0.1, 15, 146 36 GGGG TTGGGG 0.19 14 5652 TT GGGG TT GGGG TT P = S 0.1, 22, 227 37 GGGG TT 0.1819 5653 TT GGGG TT GGGG TT P = S 0.12, 27 38 GGGG 5676 GGGG TT GGGG TT P= S 0.18, 21, 114 39 GGGG 0.28 23 5675 TT GGGG TT GGGG TT P = S 0.38 1436 40 5674 TT GGGG TT GGGG P = S 0.43 >200 41 4717 GGGG TT GGGG P = S0.41 >25, 26 39 5320 TT GGGG TT P = S 0.47 195, 415 52 5739 TT GGGG P =S 3.8 −200 4803 GGGG P = S 4 >25, 13 5367 TTG GGG TTG GGG TTG P = O0.09, 52 400 34 GGG TTG GGGG 0.13 5649 TT GGGG TT GGGG TT P = O <0.08,24, 300 43 GGGG TT GGGG 0.3 31 5670 GGGG TT GGGG TT P = O 0.17, 15 44GGGG TT GGGG 5650 TT GGGG TT GGGG TT P = O 0.64 7.6 12 45 GGGG TT 5666TT GGGG TT GGGG TT P = O 0.17, 16.7, 100 54 GGGG 0.6 5 5669 GGGG TT GGGGTT P = O 1.2 9.6 9 55 GGGG 5667 TT GGGG TT GGGG TT P = O >22 5.6 56 5668TT GGGG TT GGGG P = O >21 5.2 57 5590 GGGG TT GGGG P = O >25 20 46 5671TT GGGG TT P = O 16 18, 1 15 5672 TT GGGG P = O >16 18 5673 GGGG P =O >1 43A number of compounds with significant HIV antiviral activity (I.C.₅₀ 2μM or less) were identified. Compound 5058 is a prototypicalphosphorothioate 8-mer oligonucleotide containing a G₄ core. When the G₄core was lengthened to G₅ or G₆, activity was retained. When the G₄ corewas substituted with A₄, C₄ or T₄, activity was lost. A change in thebackbone from phosphorothioate to phosphodiester also produced inactivecompounds. The oligonucleotides containing a single G₄ run were alsofound to be inactive as phosphodiesters. However, it was found thatoligonucleotides with multiple G₄ repeats are active as phosphodiesteranalogs. Substitution of the nucleotides flanking the G₄ core resultedin retention of HIV antiviral activity. The compound TTGGGGTT (ISIS5320) was the most active of the series. Compounds with 3 G's in a rowor 2 G's in a row were found to be inactive. Compounds with variousmultiples of G₄ and T₂ were generally more active than the parentTTGGGGTT. However, T₂G₄ and G₄ were less active. It was found that itwas not absolutely necessary that G₄ be flanked on both sides becauseG₄T₂G₄ is very active.Phospholipase A₂ Enzyme Activity

Specific oligonucleotide compositions having a G₄ conserved sequencehave also been identified which selectively inhibit human type IIphospholipase A₂ and type II phospholipase A₂ from selected snakevenoms. These agents may prove useful in the treatment of inflammatorydiseases, hyper-proliferative disorders, malignancies, central nervoussystem disorders such as schizophrenia, cardiovascular diseases, as wellas the sequelae resulting from the bite of poisonous snakes, mostnotably rattlesnakes.

Incubation of type II phospholipase A₂ with increasing amounts ofphosphorothioate deoxyoligonucleotides resulted in a sequence-specificinhibition of phospholipase A₂ enzyme activity. Of the oligonucleotidestested, ISIS 3196, SEQ ID NO: 47, was found to exhibit the greatestactivity, I.C.₅₀ value=0.4 μM. ISIS 3631, SEQ ID NO: 81, and 3628, SEQID NO: 78, exhibited I.C.₅₀ values approximately 10-fold higher and ISIS1573, SEQ ID NO: 120, did not significantly inhibit the phospholipase A₂at concentrations as high as 10 μM.

To further define the sequence specificity of oligonucleotides whichdirectly inhibit human type II phospholipase A₂ activity, a series ofphosphorothioate oligonucleotides were tested for direct inhibition ofenzyme activity. A compilation of the results from 43 differentsequences is shown in Table 4. TABLE 4 Sequence Specific Inhibition ofHuman Type II Phospholipase A₂ With PhosphorothioateDeoxyoligonucleotides SEQ ID ISIS # Sequence % Inhibition (1 μM) NO 3181TCTGCCCCGGCCGTCGCTCCC 42.7 58 3182 CAGAGGACTCCAGAGTTGTAT 30.2 59 3184TTCATGGTAAGAGTTCTTGGG 25.1 60 3185 CAAAGATCATGATCACTGCCA 22.7 61 3191TCCCATGQCCCTGCAGTAGGC 41.5 62 3192 GGAAGGTTTCGAGQGAAGAGG 28.1 63 3193CCTGCAGTAGGCCTGGAAGGA 22.6 64 3196 GGGTGGGTATAGAAGGGCTCC 98.5 47 3468GGGACTCAGCAACGAGGGGTG 97.5 65 3470 GTAGGGAGGGAGGGTATGAGA 88.9 66 3471AAGGAACTTGGTTAGGGTAGG 34.5 67 3472 TGGGTGAGGGATGCTTTCTGC 69.0 68 3473CTGCCTGGCCTCTAGGATGGG 25.9 69 3474 ATAGAAGGGCTCCTGCCTGGC 13.3 70 3475TCTCATTCTGGGTGGGTATAG 67.0 71 3476 GCTGGAAATCTGCTGGATGTC 43.4 72 3477GTGGAGGAGAGCAGTAGAAGG 54.7 73 3478 TGGTTAAGCACGGAGTTGAGG 26.4 74 3479CCGGAGTACAGGTTCTTTGGT 42.3 75 3480 TTGCTTTATTCAGAAGAGACC 24.5 76 3481TTTTTGATTTGCTAATTGCTT 2.2 77 3628 GGAGCCCTfCTATACCCACCC 13.6 78 3629CACCCCTCGTTGCTGAGTCCC 20.5 79 3630 TCTCATACCCTCCCTCCCTAC 17.6 80 3631AGGTCGAGGAGTGGTCTGAGC 20.7 81 3632 CCAGGAGAGGTCGGTAAGGCG 29.2 82 3633GTAGGGATGGGAGTGAAGGAG 58.5 83 3659 TGCTCCTCCTTGGTGGCTCTC 38.2 84 3663GTCTGCTGGGTGGTCTGAACT 16.3 85 3665 GGACTGGCCTAGCTCCTCTGC 45.8 86 3669GGTGACAAATGCAGATGGACT 34.7 87 3671 TAGGAGGGTCTTCATGGTAAG 49.3 88 3676AGCTCTTACCAAAGATCATGA 24.5 89 3679 AGTAGGCCTGGAAGGAAATTT 30.3 90 3688TGGCCTCACCGATCCGTTGCA 43.1 91 3694 ACAGCAGCTGTGAGGAGACAC 28.2 92 3697ACTCTTACCACAGGTGATTCT 39 93 3712 AGGAGTCCTGTTTTGAAATCA 31.8 94 4015GTTGGAGACCGGGGTTGGGG 79.4 21 4133 AGTGCACGTTGAGTATGTGAG 37.3 95 4149CTACGGCAGAGACGAGATAGC 20.2 96 4338 ACCGCCAGGGGAATCCGTCAT 100 12 4560GGGGCGGGGCGGGGCGGGG 100 42

Most of the oligonucleotides significantly inhibited phospholipase A₂enzyme activity at a concentration of 1 μM. Furthermore, a population ofoligonucleotides were found to completely inhibit phospholipase A₂activity at 1 μM concentration. A common feature of thoseoligonucleotides which inhibit greater than 50% phospholipase A₂ enzymeactivity is the occurrence of 2 or more runs of guanine residues, witheach run containing at least 3 bases. More guanine residues in the run,or more runs, resulted in more potent oligonucleotides. As an example,ISIS 3196, SEQ ID NO: 47, and ISIS 3470, SEQ ID NO: 66, both have threesets of guanine runs, with each run three bases in length. Botholigonucleotides completely inhibited human type II phospholipase A₂enzyme activity at a concentration of 1 μM. Two oligonucleotides werefound to be an exception to this finding. ISIS 3477, SEQ ID NO: 73,contained 3 sets of guanine runs, but they were only 2 bases in length.This oligonucleotide inhibited enzyme activity by 54.7% at 1 μM. Asecond oligonucleotide, ISIS 4338, SEQ ID NO: 12, contained only 1 runof guanine residues, 4 bases in length. In this experiment, ISIS 4338,SEQ ID NO: 12, completely inhibited human type II phospholipase A₂ at aconcentration of 1 μM.

To further define the minimum pharmacophore responsible for inhibitionof human type II phospholipase A₂, truncated versions of ISIS 3196, SEQID NO: 47 and 4015, SEQ ID NO: 21, were tested for activity. Inaddition, the effects of base substitutions on the activity of atruncated version of ISIS 3196, SEQ ID NO: 47, were investigated. Theresults are shown in Table 5. As the effects of base substitution andtruncation were performed in two separate experiments, the data fromboth experiments are shown. TABLE 5 Identification of the MinimumPharmacophore for PLA₂ Inhibition SEQ ISIS % Inhibition ID # Sequence (1μM) NO 3196 GGG TGG GTA TAG AAG GGC TCC 76.2 47 GGG TGG GTA TAG AAG GGC85.3 97 GGG TGG GTA TAG AAG 82.5 98 4672 GGG TGG GTA TAG 73.9 50 TGG GTATAG AAG GGC TCC 84.6 99 GTA TAG AAG GGC TCC 9.2 100 TAG AAG GGC TCC 0101 TGG GTA TAG AAG GGC 33.5 102 3196 GGG TGG GTA TAG AAG GGC TCC 100 474672 GGG TGG GTA TAG 94.6 50 4947 A GG TGG GTA TAG 22.7 103 4955 GGG AGG GTA TAG 97.5 104 4956 GGG CGG GTA TAG 92.0 105 4957 GGG TGG A TA TAG81.9 106 4946 GGG TGG G A A TAG 73.2 107 4962 GGG TGG GTA T 36.3 1084015 GTT GGA GAC CGG GGT TGG GG 98.5 21 4771 GTT GGA GAC CGG GGT TGG17.1 27 4549 GGA GAC CGG GGT TGG GG 96.2 22 4717 GG GGT TGG GG 83.1 265544 TGG GG 50 4803 GG GG

These results demonstrate that the minimum pharmacophore is 4 G's or tworuns of 3 guanines. For ISIS 4015, SEQ ID NO: 21, a 10-basephosphorothioate oligonucleotide containing the sequence GGGGTTGGGGretains full inhibitory activity. A 5-base phosphorothioateoligonucleotide with the sequence TGGGG (ISIS 5544) inhibited enzymeactivity by 50% at 1 μM; complete inhibition of enzyme activity wasobserved at a concentration of 3 μM by ISIS 5544.

A 12-base phosphorothioate oligonucleotide with the sequenceGGGTGGGTATAG (ISIS 4672, SEQ ID NO: 50) was shown in one experiment toexhibit almost the same inhibition as the 21 base oligonucleotide, ISIS3196, SEQ ID NO: 47 (Table 5). Removal of the last two 3′-bases from the12-mer results in a loss of activity (ISIS 4962, SEQ ID NO: 108). Basesubstitutions experiments demonstrate that the base separating the twoguanine runs does not markedly affect the activity. Substitution of the5′-guanine with an adenine results in loss of activity. These datasuggest that the 5′-guanine plays an important role in maintaining theactivity of the oligonucleotide. Further supporting an important role ofthe 5′-guanine in this sequence was the finding that addition of afluorescein phosphoramidite or a 5′-phosphate resulted in loss ofactivity.

All of the oligonucleotides used in the assays described above weredeoxyoligonucleotides. To determine if the effects were specific to DNAoligonucleotides, 2′-substituted analogs were tested for activity. Theresults are shown in FIG. 4. In each case the internucleosidic linkagewas phosphorothioate. No difference in potency was observed if the2′-positions were substituted with fluorine. Substitution of the2′-position with methyl and propyl enhanced the inhibitory activitytowards human type II phospholipase A₂. Replacement of thephosphorothioate backbone with phosphodiester backbone resulted in lossof inhibitory activity. This loss of inhibitory activity byphosphodiester oligonucleotides was not due to degradation of theoligonucleotides, as the oligonucleotides were found to be stable for atleast 4 hours in the incubation buffer. The phospholipase A₂ enzymeassays were 15 minutes in duration.

In summary, these results demonstrate that phosphorothioateoligonucleotides containing two or more runs of guanines, with each runat least three bases in length are potent inhibitors of human type IIphospholipase A₂ enzyme activity. Substitution of the 2′-position witheither methyl or propyl groups enhanced inhibitory activity. Thephosphorothioate internucleosidic linkage was found to be obligatory forbiological activity.

Modulation of Telomere Length

Oligonucleotides capable of modulating telomere length are alsocontemplated by this invention. In human cells, the sequence TTAGGG isrepeated from hundreds to thousands of times at both ends of everychromosome, depending on cell type and age. It is believed thatoligonucleotides having a sequence (N_(X)G₃₋₄)_(Q)N_(X) wherein X is 1-8and Q is 1-6 would be useful for modulating telomere length.

Since telomeres appear to have a role in cell aging, i.e., telomerelength decreases with each cell division, it is believed that sucholigonucleotides would be useful for modulating the cell's agingprocess. Altered telomeres are also found in cancerous cells; it istherefore also believed that such oligonucleotides would be useful forcontrolling malignant cell growth. Therefore, modulation of telomerelength using oligonucleotides of the present invention could proveuseful for the treatment of cancer or in controlling the aging process.

The following examples are provided for illustrative purposes only andare not intended to limit the invention.

EXAMPLES Example 1 Oligonucleotide Synthesis

DNA synthesizer reagents, controlled-pore glass (CPG)-bound andB-cyanoethyldiisopropylphosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). 2′-O-MethylB-cyanoethyldiisopropylphosphoramidites were purchased from Chemgenes(Needham, Mass.). Phenoxyacetyl-protected phosphoramadites for RNAsynthesis were purchased from BioGenex (Hayward, Calif.).

Oligonucleotides were synthesized on an automated DNA synthesizer(Applied Biosystems model 380B). 2′-O-Methyl oligonucleotides weresynthesized using the standard cycle for unmodified oligonucleotides,except the wait step after pulse delivery of tetrazole and base wasincreased to 360 seconds. The 3′ base bound to the CPG used to start thesynthesis was a 2′-deoxyribonucleotide. After cleavage from the CPGcolumn and deblocking in concentrated ammonium hydroxide at 55° C. (18hours), the oligonucleotides were purified by precipitation two timesout of 0.5 M NaCl solution with 2.5 volumes ethanol. Analytical gelelectrophoresis was accomplished in 20% acrylamide, 8 M urea, 45 mMTris-borate buffer, pH=7.0. Oligonucleotides were judged frompolyacrylamide gel electrophoresis to be greater than 85% full lengthmaterial.

Example 2 HIV Inhibition Acute HIV Infection Assay

The human T-lymphoblastoid CEM cell line was maintained in exponentialgrowth phase in RPMI 1640 with 10% fetal calf serum, glutamine, andantibiotics. On the day of the assay, the cells were washed and countedby trypan blue exclusion. These cells (CEM-IIIB) were seeded in eachwell of a 96-well microtiter plate at 5×10³ cells per well. Followingthe addition of cells to each well, the oligonucleotides were added atthe indicated concentrations and serial half log dilutions. InfectiousHIV-1_(IIIB) was immediately added to each well at a multiplicity ofinfection determined to give complete cell killing at 6 dayspost-infection. Following 6 days of incubation at 37° C., an aliquot ofsupernatant was removed from each well prior to the addition of thetetrazolium dye XTT to each well. The XTT was metabolized to a formazanproduct by viable cells and the results calculatedspectrophotometrically with a Molecular Devices Vmax Plate Reader. TheXTT assay measures protection from the HIV-induced cell killing as aresult of the addition of test compounds. The supernatant aliquot wasutilized to confirm the activities determined in the XTT assay. Reversetranscriptase assays and p24 ELISA were performed to measure the amountof HIV released from the infected cells. Protection from killing resultsin an increased optical density in the XTT assay and reduced levels ofviral reverse transcriptase and p24 core protein.

Example 3 HSV-1 Inhibition HSV-1 Infection ELISA Assay

Confluent monolayers of human dermal fibroblasts were infected withHSV-1 (KOS) at a multiplicity of 0.05 pfu/cell. After a 90 minuteadsorption at 37° C., virus was removed and culture medium containingoligonucleotide at the indicated concentrations was added. Two daysafter infection medium was removed and cells fixed by addition of 95%ethanol. HSV antigen expression was quantitated using an enzyme linkedimmunoassay. Primary reactive antibody in the assay was a monoclonalantibody specific for HSV-1 glycoprotein B. Detection was achieved usingbiotinylated goat anti-mouse IgG as secondary antibody followed byreaction with streptavidin conjugated B-galactosidase. Color wasdeveloped by addition of chlorophenol red B-D-galactopyranoside andabsorbance at 570 nanometers was measured. Results are expressed aspercent of untreated control.

Virus Yield Assay.

Confluent monolayers of human dermal fibroblasts were infected withHSV-1 (KOS) at a multiplicity of 0.5 pfu/cell. After a 90 minuteadsorption at 37° C., virus was removed and 1 ml of culture mediumcontaining oligonucleotide at the indicated concentrations was added.Control wells received 1 ml of medium which contained nooligonucleotide. 2 days after infection, culture medium and cells wereharvested and duplicate wells of each experimental point were combined.The suspension was frozen and thawed 3 times, then drawn through a 22gauge needle five times. Virus titer was determined by plaque assay onVero cell monolayers. Dilutions of each virus preparation were preparedand duplicates were adsorbed onto confluent Vero monolayers for 90minutes. After adsorption, virus was removed, cells were rinsed oncewith phosphate-buffered saline, and overlaid with 2 ml of mediumcontaining 5.0% FBS and methyl cellulose. Cells were incubated at 37° C.for 72 hours before plaques were fixed with formaldehyde and stainedwith crystal violet. The number of plaques from treated wells wascompared to the number of plaques from control wells. Results areexpressed as percent of virus titer from untreated control cells andshown in FIG. 2.

Example 4 Cytomegalovirus Inhibition ELISA Assay

Confluent monolayer cultures of human dermal fibroblasts were treatedwith oligonucleotides at the indicated concentrations in serum-freefibroblast growth medium. After overnight incubation at 37° C., culturemedium containing oligonucleotides was removed, cells were rinsed andhuman cytomegalovirus was added at a multiplicity of infection of 0.1pfu/cell. After a 2 hour adsorption at 37° C., virus was removed andfresh fibroblast growth medium containing oligonucleotide at theindicated concentrations was added. Two days after infection, oldculture medium was removed and replaced with fresh fibroblast growthmedium containing oligonucleotides at the indicated concentrations. Sixdays after infection media was removed, and cells fixed by addition of95% ethanol. HCMV antigen expression was quantitated using an enzymelinked immunoassay. Primary reactive antibody in the assay was amonoclonal antibody specific for a late HCMV viral protein. Detectionwas achieved using biotinylated goat anti-mouse IgG as secondaryantibody followed by reaction with streptavidin conjugatedB-galactosidase. Color was developed by addition of chlorophenol redB-D-galactopyranoside and absorbance at 575 nanometers measured using anELISA plate reader. Results are expressed as percent of untreatedcontrol.

Example 5 Influenza Virus Inhibition Virus Yield Assay

Confluent monolayer cultures of Madin-Darby canine kidney (MDCK) cellswere treated with oligonucleotide at a concentration of 10 mM inserum-free Dulbecco's modified Eagle's medium (DMEM) containing 0.2%BSA. After incubation at 37° C. for 2 hours, human influenza virus (A/PRstrain) was added to the cells at a multiplicity of infection of 0.00125pfu/cell. Virus was adsorbed for 30 minutes at 37° C. Cells were washedand refed with fresh medium containing oligonucleotide at aconcentration of 10 μM, plus 0.2% BSA, and 3 mg/ml trypsin. One dayafter infection, medium was harvested. Viral supernatants were titeredon MDCK cells. MDCK cells grown in 6-well dishes were infected withdilutions of each virus preparation. After adsorption for 30 minutes at37° C., virus was removed from the monolayers and cells were overlaidwith 2.5 ml of fresh medium containing 0.2% BSA, 3 μg/ml trypsin, and0.44% agarose. Twenty-four hours after infection, cells were fixed in3.5% formaldehyde and plaques visualized by staining monolayers withcrystal violet. Results are expressed as a percentage of the titer ofvirus stock from untreated MDCK cells.

Example 6 Identification of Oligonucleotide Inhibition of Human Type IIPhospholipase A₂

The human epidermal carcinoma cell line A431 was purchased from AmericanType Culture Collection. Cells were grown in Dulbecco's Modified Eagle'sMedium containing 4.5 gm glucose per liter and 10% fetal calf serum.Type II phospholipase A₂ was prepared from A431 cells by cultivatingconfluent monolayers with Opti-MEM (Gibco). The medium was concentrated5 to 10 fold on an Amicon ultrafiltration device using YM-5 membranes.The concentrated spent medium was used as a source of human type IIphospholipase A₂. Previous studies have demonstrated that A431 cellsonly secrete type II phospholipase A₂.

Phospholipase A₂ assays were performed utilizing ³H-oleic acid labelledE. coli as the substrate. ³H-Oleic acid labelled E. coli were preparedas described by Davidson et al. J. Biol. Chem. 1987, 262, 1698). Thereactions contained 100,000 cpm of ³H-oleic acid labelled E. coli, 50 mMTris-HCl, pH=7.4, 50 mM NaCl, 1 mM CaCl₂, and 50 μg bovine serum albuminin a final reaction volume of 200 μL. Reactions were initiated by theaddition of the E. coli substrate. Reactions were terminated by theaddition of 100 μL 2 N HCl and 100 μL 100 mg/ml fatty acid free bovineserum albumin. Samples were vortexed and centrifuged at 17,000×g for 5minutes. The amount of ³H-oleic acid in the supernatant was determinedby counting a 300 μL aliquot in a liquid scintillation counter.Oligonucleotides were added to the incubation mixture prior to theaddition of the substrate.

Example 7 Structural Requirement for Inhibition of Human Type IIPhospholipase A₂ by Phosphorothioate Oligonucleotides

The oligonucleotides which inhibit human type II phospholipase A₂ sharea common feature with telomeric DNA sequences in that both are composedof guanine rich sequences. Telomeric sequences such as that fromOxytricha (XXXG₄T₄G₄T₄G₄T₄G₄T₄G₄, SEQ ID NO: 121) form an unusualstructure termed a G quartet. The formation of this structure ismonovalent cation dependent and is disrupted by high temperature. Todetermine if oligonucleotide structure was part of the activepharmacophore, ISIS 3196, SEQ ID NO: 47, was placed in boiling water for15 minutes prior to addition to the assay. Boiling reduced theinhibitory activity of ISIS 3196, SEQ ID NO: 47, from 94% inhibition to21% inhibition. Examination of the oligonucleotide by denaturing gelelectrophoresis demonstrated that boiling did not cause theoligonucleotide to fragment. Separation of native and denatured ISIS3196, SEQ ID NO: 47, by gel filtration chromatography on a Superdex G-75column demonstrated that in its native conformation, thisoligonucleotide exists as several molecular species. Boiling ISIS 3196,SEQ ID NO: 47, prior to chromatography resulted in loss of highmolecular weight species and appearance of the oligonucleotide in thelower molecular weight species. From these studies we can conclude thatstructure appears to be part of the pharmacophore for ISIS 3196, SEQ IDNO: 47.

Example 8 Specificity of Phosphorothioate Oligonucleotide for SelectType II Phospholipase A₂

Bovine pancreatic phospholipase A₂ , Apis mellifera phospholipase A₂ ,Naja naja naja phospholipase A₂, and Crotalus durissus terrificusphospholipase A₂ were obtained from Sigma Chemical Co. (St. Louis, Mo.).Phospholipase A₂ isolated from the venom of Trimeresurus flavoridis wasobtained from Calbiochem (La Jolla, Calif.), and phospholipase A₂ fromAgkistrodon piscivorus piscivorus was partially purified from wholevenom (Sigma Chemical Co.) by chromatography on a Mono S column(Pharmacia, Upsalla, Sweden).

To determine the specificity of ISIS 3196, SEQ ID NO: 47, towards humantype II phospholipase A₂, phospholipase A₂ from different sources weretested for inhibitory activity (FIG. 5). Human type II phospholipase A₂was the most sensitive of all the enzymes tested to the inhibitoryeffects of ISIS 3196, SEQ ID NO: 47, I.C.₅₀≈0.15 μM (FIG. 5).Phospholipase A₂ isolated from Crotalus durissus venom (rattlesnake),also a type II enzyme, was the next most sensitive to the effects ofISIS 3196, SEQ ID NO: 47, I.C.₅₀≈0.3 μM, followed by phospholipase A₂isolated from the venom of Agkistrodon piscivorus piscivorus(cottonmouth), also a type II enzyme, I.C.₅₀≈3 μM. Bovine pancreaticphospholipase A₂, a type I enzyme, was the most resistant of all theenzymes tested to the effects of ISIS 3196, SEQ ID NO: 47, I.C.₅₀≈100 μM(FIG. 5). Phospholipase A₂ isolated from Naja naja naja venom (cobravenom), a type 1 enzyme and from Trimeresurus flavoridis (Asian pitviper, habu) were both relatively resistant to the inhibitory effect ofISIS 3196, SEQ ID No; 47, with I.C.₅₀ values greater than 10 μM.Phospholipase A₂ isolated from Apis mellifera (honeybee), neither a typeI or type II enzyme, was also quite resistant to the inhibitory activityof ISIS 3196, SEQ ID NO: 47, with an I.C.₅₀ value greater than 100 μM.

These results demonstrate that ISIS 3196, SEQ ID NO: 47, selectivelyinhibits human type II phospholipase A₂. Other type II phospholipase A₂,such as those isolated from Crotalus and Agkistrodon venoms, were alsosensitive to the effects of ISIS 3196, SEQ ID NO: 47. While, in general,type I enzymes were more resistant to the effects of ISIS 3196, SEQ IDNO: 47. Although bee venom (Apis mellifera) phospholipase A₂ does notbear a strong sequence homology to either type I or type II enzymes, itis more closely related to type I enzymes. Like other type I enzymes, itis relatively resistant to the inhibitor effects of ISIS 3196, SEQ IDNO: 47.

Example 9 Mechanism of Inhibition of Human Type II Phospholipase A₂ byPhosphorothioate Oligonucleotides

As a first step in elucidation of the mechanism by whichphosphorothioate oligonucleotides inhibit phospholipase A₂, the effectsof the oligonucleotides on the substrate kinetics of the enzymes weredetermined. Human type II phospholipase A₂ was incubated with increasingamounts of E. coli substrate in the presence of oligonucleotides ISIS3196, SEQ ID NO: 47, and ISIS 3481, SEQ ID NO: 77 (FIG. 6). Theconcentration of E. coli phospholipid was determined by lipid phosphorusanalysis as described by Bartlett, J. Biol. Chem. 1959, 234:466. Theresults demonstrate that ISIS 3481, SEQ ID NO: 77, at 0.2 μM and 2 μMdid not modify the substrate kinetics of human type II phospholipase A₂.In contrast, ISIS 3196, SEQ ID NO: 47, behaved as an apparentnoncompetitive inhibitor in that the apparent Km and Vmax were bothchanged in the presence of the oligonucleotide. It is unlikely that ISIS3196, SEQ ID NO: 47, inhibits human type H phospholipase A₂ by chelatingcalcium which is required for activity, in that the free calcium in theassay was in 500 to 5000-fold excess to the oligonucleotide.

Example 10 Modulation of Telomere Length by G₄ PhosphorothioateOligonucleotides

The amount and length of telomeric DNA in human fibroblasts has beenshown to decrease during aging as a function of serial passage in vitro.To examine the effect of G₄ phosphorothioate oligonucleotides on thisprocess, human skin biopsy fibroblasts are grown as described in Harley,C. B., Meth. Molec. Biol. 1990, 5, 25-32. Cells are treated with theoligonucleotides shown in Table 6, by adding the oligonucleotide to themedium to give a final concentration of 1 μM, 3 μM or 10 μM; controlcells receive no oligonucleotide. Population doublings are counted andDNA is isolated at regular intervals. Telomere length is determined bySouthern blot analysis and plotted against number of populationdoublings as described in Harley, C. B. et al., Nature 1990, 345,458-460. The slope of the resulting linear regression lines indicates aloss of approximately 50 bp of telomere DNA per mean population doublingin untreated fibroblasts. Harley, C. B. et al., Nature 1990, 345,458-460. Treatment with oligonucleotides of Table 6 is expected toresult in modulation of telomere length. TABLE 6 Effect of G₄Phosphorothioate Oligonucleotides on Telomere Length in AgingFibroblasts ISIS NO. SEQUENCE SEQ ID NO: TT AGGG 5739 TT GGGG 5756 TTAGGG TT 5320 TT GGGG TT 5675 TT GGGG TT GGGG TT 40 5651 TT GGGG TT GGGGTT GGGG TT GGGG 35 TTTT GGGG TTTA GGGG 5673 GGGG

Example 11 Activity of G₄ Phosphorothioate Oligonucleotides AgainstSeveral Viruses

Antiviral activity of oligonucleotides was determined by CPE inhibitionassay for influenza virus, adenovirus, respiratory syncytial virus,human rhinovirus, vaccinia virus, HSV-2 and varicella zoster virus. TheMTT cell viability assay was used to assay effects on HIV. HSV-2,adenovirus, vaccinia virus and rhinovirus were assayed in MA104 cells.Respiratory syncytial virus was assayed in HEp-2 cells and influenzavirus was assayed in MDCK cells. CEM cells were used in MTT assays ofHIV inhibition. Oligonucleotide was added at time of virus infection.

MDCK (normal canine kidney) cells and HEp-2, a continuous humanepidermoid carcinoma cell line, were obtained from the American TypeCulture Collection, Rockville, Md. MA-104, a continuous line of Africangreen monkey kidney cells, was obtained from Whittaker M. A.Bioproducts, Walkersville, Md.

HSV-2 strain E194 and influenza strain A/NWS/33 (H1N1) were used.Adenovirus, Type 5 (A-5), strain Adenoid 75; respiratory syncytial virus(RSV) strain Long; rhinovirus 2 (R-2), strain HGP; and vaccinia virus,stain Lederle-chorioallantoic were obtained from the American TypeCulture Collection, Rockville Md.

Cells were grown in Eagle's minimum essential medium with non-essentialamino acids (MEM, GIBCO-BRL, Grand Island N.Y.) with 9% fetal bovineserum (FBS, Hyclone Laboratories, Logan Utah), 0.1% NaHCO₃ for MA104cells; MEM 5% FBS, 0.1% NaHCO₃ for MDCK cells, and MEM, 10% FBS, 0.2%NaHCO₃ for HEp-2 cells. Test medium for HSV-2, A-5, R-2 and vacciniavirus dilution was MEM, 2% FBS, 0.18% NaHCO₃, 50 μg gentamicin/ml. RSVwas diluted in MEM, 5% FBS, 0.18% NaHCO₃, 50 μg gentamicin/ml. Testmedium for dilution of influenza virus was MEM without serum, with 0.18%NaHCO₃, 20 μg trypsin/ml, 2.0 μg EDTA/ml, 50 μg gentamicin/ml.

Ribavirin was obtained from ICN Pharmaceuticals, Costa Mesa, Calif.Acyclovir and 9β-D-arabinofuranosyladenine (ara-A) were purchased fromSigma Chemical Co., St. Louis, Mo. Ribavirin, acyclovir and ara-A wereprepared and diluted in MEM without serum, plus 0.18% NaHCO₃, 50 μggentamicin/ml. Oligonucleotides were diluted in the same solution.

Cells were seeded in 96-well flat bottom tissue culture plates, 0.2ml/well, and incubated overnight in order to establish monolayers ofcells. Growth medium was decanted from the plates. Compound dilutionswere added to wells of the plate (4 wells/dilution, 0.1 ml/well for eachcompound) as stocks having twice the desired final concentration.Compound diluent medium was added to cell and virus control wells (0.1ml/well). Virus, diluted in the specified test medium, was added to allcompound test wells 3 wells/dilution) and to virus control wells at 0.1ml/well. Test medium without virus was added to all toxicity controlwells (1 well/dilution for each comopund test) and to cell control wellsat 0.1 ml/well. The plates were incubated at 37° C. in a humidifiedincubator with 5% CO₂, 95% air atmosphere until virus control wells hadadequate CPE readings. Cells in test and virus control wells were thenexamined microscopically and graded for morphological changes due tocytotoxicity. Effective dose, 50% endpoint (ED50) and cytotoxic dose,50% endpoint (CD50) were calculated by regression analysis of the viralCPE data and the toxicity control data, respectively. The ED50 is thatconcentration of compound which is calculated to produce a CPE gradehalfway between that of the cell controls (0) and that of the viruscontrols. CD50 is that concentration of compound calculated to behalfway between the concentration which produces no visible effect onthe cells and the concentration which produces complete cytotoxicity.The therapeutic index (TI) for each substance was calculated by theformula: TI=CD50/ED50.

Oligonucleotide sequences are shown in Table 1 except for ISIS 3383 (SEQID NO: 122) and ISIS 6071. ISIS 3383 is a scrambled version of ISIS 1082(SEQ ID NO: 134). ISIS 6071 (TGTGTGTG) is a scrambled version of ISIS5320. The results are shown in Table 7. Oligonucleotides with ED50values of less than 50 μM were judged to be active in this assay and arepreferred. TABLE 7 Oligonucleotide activity against RNA and DNA virusesVirus: DNA Viruses RNA Viruses Compound: HSV-2 VZV A-5 Vacc RSV RhinoHIV Influenza 3383 ED50 2.8 μM — >100 >100 0.7 >100 — 19 TI >36 — — — 60— — >5 4015 ED50 0.8 29 >100 15 0.6 >100 0.16 0.6 TI >125 1.0 <1.0 >6.793 — 100 93 3657 ED50 0.6 >100 >100 18 0.8 >100 — 1.0 TI >167 1.0<1.0 >5.6 >125 — — 56 4338 ED50 0.6 — 68 19 1.0 >100 — 0.5 TI >53— >1.5 >5.3 13 — — >200 1220 ED50 0.7 — >50 46 — >50 — — TI >71 — — >1.1— — — — 5652 ED50 0.3 18 >100 — 1.9 >100 0.18 0.6 TI >333 — <1.0 — >53 —227 93 ACV ED50 97.7 — — — — — — — TI >45 — — — — — — — Ribavirin ED50 —— 82 — 49 229 — 7.78 TI — — 28 — 20 10 — 202 Ara-A ED50 — — — 15.8 — — —— TI — — — 125 — — — — 5320 ED50 4 >100 >100 >100 — — 0.4 40 TI — — — —— — 390 — 6071 ED50 >100 >100 >100 >100 — — 50 >100 TI — — — — — — — —

Example 12 Testing of Oligonucleotides for Activity Against HSV-1

Phosphorothioate oligonucleotides were synthesized which arecomplementary to regions of the HSV-1 RNA containing clusters ofcytosines. These oligonucleotides are shown in Table 8: TABLE 8Phosphorothioate oligonucleotides targeted to HSV-1 (sequences written5′ TO 3′) SEQ Oligo Target ID # Sequence Target Function NO: 1220 CACGAA AGG CAT UL9, Ori binding 1 GAC CGG GGC AUG protein 4274 CAT GGC GGGACT UL27, virion gB 8 ACG GGG GCC AUG 4338 ACC GCC AGG GGA UL42, DNAbinding 12 ATC CGT CAT AUG protein 4346 GAG GTG GGC TTC UL42, ″ 123 GGTGGT GA 5′UTR 3657 CAT CGC CGA TGC IE175, Transc. 16 GGG GCG ATC AUGtransactivator 4015 GTT GGA GAC CGG UL29, ssDNA 21 GGT TGG GG 5′UTRbinding protein 4398 CAC GGG GTC GCC ″ ″ 28 GAT GAA CC 4393 GGG GTT GGGGAA ″ ″ 124 TGA ATC CC 4348 GGG TTG GAG ACC ″ ″ 125 GGG GTT GG 4349 GGTTGG AGA CCG ″ ″ 126 GGG TTG GG 4341 TGG AGA CCG GGG ″ ″ 127 TTG GGG AA4342 TTG GAG ACC GGG ″ ″ 128 GTT GGG GA 4350 GAC GGT CAA GGG ″ ″ 129 GAGGGT TGG 4435 GGG GAG ACC GAA UL20, Viral egress 130 ACC GCA AA 5′UTR4111 CCT GGA TGA TGC UL30, DNA polymerase 131 TGG GGT AC coding 4112 GACTGG GGC GAG ″ ″ 132 GTA GGG GT 4399 GTC CCG ACT GGG ″ ″ 133 GCG AGG AT

The oligonucleotides shown in Table 8 were tested for activity againstHSV-1 (KOS strain) using an ELISA assay as described in Example 3.Results are expressed as percent of untreated control. From theseresults, an EC50 (effective oligonucleotide concentration giving 50%inhibition) is calculated for each oligonucleotide. These values,expressed in μM, are given in Table 9. Oligonucleotides having EC50s of1 μM or less in this ELISA assay were judged to have particularly goodactivity and are preferred. The negative control oligonucleotide, ISIS1082 (complementary to HSV UL13 translation initiation codon; has noruns of G) had EC50 of 2.5 and 1.8 μM in duplicate experiments. TABLE 9Oligonucleotide inhibition of HSV-1 All oligonucleotides arephosphorothioates Oligo # EC50 (μM)* 1220 0.24, 0.16 4274 0.15, 0.154338 0.20, 0.20 4346 0.50 3657 0.20 4015 0.22, 0.22 4398 0.10 4393 0.204348 0.40 4349 0.25 4341 0.20 4342 0.20 4350 0.25 4435 0.22 4111 0.604112 0.30 4399 0.25*Some experiments were done in duplicate

Example 13 Activity of G₄ Phosphorothioate Oligonucleotides AgainstVarious Strains of HSV

Oligonucleotides were tested against HSV-1 and five strains of HSV-1, ofwhich two (HSV1-DM2.1 and HSV1-PAAr) are resistant to acyclovir (ACV).Oligonucleotides were assayed by ELISA as described in Example 3 andresults are shown in Table 10. In this assay, oligonucleotides withEC50s of 1 μM or less were judged to be particularly active and arepreferred. TABLE 10 Oligonucleotide activity against various HSV strainsResults are given as EC50, expressed in μM Compound: 4015 1220 3657 43384274 1082 ACV SEQ ID NO: HSV strain 21 1 16 12 8 134 HSV-1 (KOS) 0.250.34 0.38 0.24 0.21 2.1 2.5 HSV-2 0.2 0.1 0.2 0.2 0.2 2.0 2.0 HSV1-F0.22 0.22 0.22 0.25 0.25 >3.0 0.7 HSV1-McKrae 0.45 0.30 0.40 0.60 >3.01.8 HSV1-DM2.1 0.10 0.10 0.10 0.70 0.40 >3.0 >3.0 HSV1-PAAr 0.35 0.120.10 0.30 0.25 >3.0 >3.0

Example 14 Effect of Time of Oligonucleotide Addition on HSV-1Inhibition by G₄ Phosphorothioate Oligonucleotides

NHDF cells were infected with HSV-1 (KOS) at a MOI of 3.0 pfu/cell.Oligonucleotides or ACV were added at a concentration of 12 mM atdifferent times after infection. HSV was detected by ELISA 48 hoursafter infection. It was found that all oligonucleotides, includingscrambled control oligonucleotide 3383, inhibited HSV replication whenadded to cells at the time of virus infection (t=0), but onlyoligonucleotides complementary to HSV genes (ISIS 4274, 1220, 4015 and3657) inhibited HSV replication when added after virus infection.Oligonucleotides showed good antiviral activity when added 8 to 11 hoursafter infection. This pattern is similar to that observed with ACV, asshown in FIG. 7.

Example 15 Chimeric 2′-O-methyl G₄ Oligonucleotides with Deoxy Gaps

A series of phosphorothioate oligonucleotides were synthesized having a2′-O-methyl substitution on the sugar of each nucleotide in the flankingregions, and 2′-deoxynucleotides in the center portion of theoligonucleotide (referred to as the “deoxy gap”). Deoxy gaps varied fromzero to seven nucleotides in length. These chimeric oligonucleotideswere assayed by ELISA as described in Example 3 and results are shown inTable 11. In this assay, oligonucleotides with EC50s of 1 μM or lesswere judged to be particularly active and are preferred. TABLE 11Activity of 2′-O-me C₄ oligonucleotides against HSV (2′-O-me nucleotidesshown in bold) SEQ Oligo EC50 ID # Sequence Target Type (μM) NO: 1220CAC GAA AGG CAT GAC UL9, Parent 0.24, 1 CGG GGC AUG (deoxy) 0.16 4240CAC GAA AGG CAT GAC ″ Deoxy 1 CGG GGC gap 3657 CAT CGC CGA TGC GGGIE175, Parent 0.20 16 GCG ATC AUG (deoxy) 5377 CAT CGC CGA TGC GGG ″2′-O-me 1.20 16 GCG ATC 4237 CAT CGC CGA TGC GGG ″ Deoxy gap 16 GCG ATC4015 GTT GGA GAC CGG GGT UL29, Parent 0.22, 21 TGG GG 5′UTR (deoxy) 0.224538 GTT GGA GAC CGG GGT ″ Deoxy gap 0.16 21 TGG GG 5378GTT GGA GAC CGG GGT ″ 2′-O-me 0.40 21 TGG GG 4398 CAC GGG GTC GCC GATUL29, Parent 0.10 28 GAA CC 5′UTR (deoxy) 5039 CAC GGG GTC GCC GAT ″2′-O-me 2.70 28 GAA CC 5189 CAC GGG GTC GCC GAT ″ Deoxy gap 0.16 28GAA CC

Additional chimeric oligonucleotides were synthesized having thesequences of ISIS 4015 and ISIS 4398. These oligonucleotides were2′-O-methyl oligonucleotides with deoxy gaps as described above, butinstead of a uniform phosphorothioate backbone, these compounds hadphosphorothioate internucleotide linkages in the deoxy gap region andphosphodiester linkages in the flanking region. These oligonucleotideswere not active against HSV in this ELISA assay.

Additional oligonucleotides were synthesized with 2′-O-propylmodifications. 2′-O-propyl oligonucleotides were prepared from2′-deoxy-2′-O-propyl ribosides of nucleic acid bases A, G, U(T), and Cwhich were prepared by modifications of literature procedures describedby B. S. Sproat, et al., Nucleic Acids Research 18:41-49 (1990) and H.Inoue, et al., Nucleic Acids Research 15:6131-6148 (1987). ISIS 7114 isa phosphorothioate which has the same sequence (SEQ ID NO: 21) as ISIS4015, and has a 2′-O-propyl modification on each sugar. ISIS 7171 is aphosphorothioate gapped 2′-O-propyl oligonucleotide with the samesequence as ISIS 4015 and 2′-O-propyl modifications at positions 1-7 and14-20 (6-deoxy gap). As shown in FIG. 8, all three oligonucleotides areactive against HSV. A uniform 2′-O-propyl phosphorothioate version ofISIS 3657 (SEQ ID NO: 16) was also synthesized and tested for activityagainst HSV-1. As shown in FIG. 9, this oligonucleotide (ISIS 7115) waseven more active than ISIS 3657. 2′-O-propyl modifications are thereforea preferred embodiment of this invention. FIG. 9 also shows that bothISIS 3657 and ISIS 7115 are several-fold more active than Acyclovir,which in turn is more active than a control oligonucleotide, ISIS 3383.

Example 16 Effect of Chemical Modification on Inhibition of HSV-1 by G₄Oligonucleotides

Inosine Substitutions:

A series of oligonucleotides were prepared in which one or moreguanosines were replaced with an inosine residue. Oligonucleotidescontaining inosine residues were synthesized as for unmodified DNAoligonucleotides, using inosine phosphoramidites purchased from GlenResearch. These sequences were assayed for activity in ELISA assays asdescribed in Example 3. These oligonucleotides, their parent sequencesand EC50 values are shown in Table 12. TABLE 12 Activity ofinosine-substituted oligonucleotides against HSV SEQ Oligo EC50 ID #Sequence Target Type (μM) NO: 1220 CAC GAA AGG CAT GAC UL9, Parent 0.24,1 CGG GGC AUG 0.16 5297 CAC GAA AGG CAT GAC ″ Inosine >3.0 135 CGI GGC#18 5308 CAC GAA AGG CAT GAC ″ Inosine >3.0 136 CGG GIC #20 4015 GTT GGAGAC CGG GGT UL29, Parent 0.22, 21 TGG GG 5′UTR 0.22 4925 GTT GGA GAC CGGIGT ″ Inosine 1.60 137 TGG IG #13, 19 5295 GTT GGA GAC CGG GIT ″Inosine >3.0 138 TGG GG #14 5296 GTT GGA GAC CGG GGT ″ Inosine 0.80 139TGG IG #19 5309 GTT GGA GAC CGI GGT ″ Inosine >3.0 140 TGG GG #12 5310GTT GGA GAC CGG GGT ″ Inosine 0.40 141 TGG GI #20

In this assay, oligonucleotides with EC50s of 1 μM or less were judgedto be particularly active and are preferred.

Fluorescein-Conjugated Oligonucleotides:

Several oligonucleotides were synthesized with a fluorescein moietyconjugated to the 5′ end of the oligonucleotide. Fluorescein-conjugatedoligonucleotides were synthesized using fluorescein-labeled amiditespurchased from Glen Research.

These sequences were assayed for activity in ELISA assays as describedin Example 3. These oligonucleotides, their parent sequences and EC50values are shown in Table 13. In this assay, oligonucleotides with EC50sof 1 μM or less were judged to be particularly active and are preferred.TABLE 13 Activity of fluorescein-conjugated oligonucleotides against HSVSEQ Oligo EC50 ID # Sequence Target Type (μM) NO: 1220 CAC GAA AGG CATUL9, Parent 0.24, 1 GAC CGG GGC AUG 0.16 5338 CAC GAA AGG CAT ″Fluorescein 0.16 1 GAC CGG GGC 3657 CAT CGC CGA TGC IE175, Parent 0.2016 GGG GCG ATC AUG 5340 CAT CGC CGA TGC ″ Fluorescein 0.18 16 GGG GCGATC 4398 CAC GGG GTC GCC UL29, Parent 0.10 28 GAT GAA CC 5′UTR 5324 CACGGG GTC GCC ″ Fluorescein 0.16 28 GAT GAA CC 1082 GCC GAG GTC CAT UL13,Parent 2.50, 134 GTC GTA CGC AUG 1.80 5339 GCC GAG GTC CAT ″ Fluorescein0.65 134 GTC GTA CGC7-Methyl-7-deaza Guanosine Substitutions:

Monomer Preparation:

A stirred suspension of 0.8 g (20 mmole) of a 60% sodium hydride inhexane dispersion was decanted and taken to dryness, resuspended in 100ml of dry acetonitrile and the suspension treated with 3.21 g (15 mmole)of 4-chloro-5-methyl-2-methylthiopyrrolo[2,3-d]pyrimidine [Kondo et al.(1977) Agric. Biol. Chem. 4:1501-1507. The mixture was stirred undernitrogen at room temperature for one hour and then treated with 5.9 g(15 mmole) of1-chloro-2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythropentofuranose added inportions. An additional 40 ml of acetonitrile was added, the mixturestirred at 50° C. for about three and one half hours and then filteredand the solid washed with acetonitrile and dried to give 6.1 g (72%) of4-chloro-5-methyl-2-methylthio-7-[α-D-erythro-pentofuranosyl]pyrrolo[2,3-d]pyrimidine,m.p. 163-163.5° C.

Reaction of this product with sodium 2-propenyloxide in DMF afforded5-methyl-2-methylthio-4-(2-propenyloxy)-7-(α-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine,which on oxidation with two molar equivalents of 3-chloroperbenzoic acidin methylene chloroide, afforded5-methyl-2-methylsulfonyl-4-(2-propenyloxy-7-(α-D-erythro-pentofuranosyl)pyrrolo[2,3-d]-pyrimidine.Reaction of the product with hydrazine afforded5-methyl-2-hydrazino-4-(2-propenyloxy)-7-(α-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine.Reduction of the product with, for example, Raney nickel affords7-deaza-2′-deoxy-7-methylguanosine.

Protection of Monomer:

The latter is treated sequentially first with trimethylchlorosilane inthe presence of pyridine, then with isobutyric hydroxide to give2-isobutyryl-7-deaza-2′-deoxy-7-methylguanosie, which, on reaction withone molar equivalent of trityl chloride in the presence of dry pyridine,affords 2-isobutyryl-7-deaza-2′-deoxy-7-methyl-5′tritylguanosine.Reaction of the latter with one molar equivalent ofchloro-β-cyanoethoxy-N,N-diisopropylaminophosphine affords2-isobutyryl-7-deaza-2′-deoxy-7-methyl-3′-O-[N,N-diisopropylamino)-β-cyanoethoxyphosphanyl]-5′-tritylguanosine.This protected monomer is then incorporated into oligonucleotides duringautomated synthesis.

An oligonucleotide having the same sequence as ISIS 3657 was synthesizedin which the guanosines at positions 14 and 15 were replaced with7-methyl-7-deaza guanosines. This oligonucleotide (ISIS 6303) was foundto have an IC50 of approximately 10 μM.

Example 17 Activity of ISIS 4015 in Combination with Other AntiviralDrugs

ISIS 4015 was tested in combination with the nucleoside analog5-trifluoromethyl-dUrd (TFT) in the ELISA assay described in Example 3.Oligonucleotide and TFT concentrations from 0 to 2 μM were tested. Asshown in FIG. 10, ISIS 4015 appears to enhance the activity of TFTagainst HSV-1.

ISIS 4015 was tested in the same way against9-(2-hydroxyethoxymethyl)guanine (Acyclovir, ACV), at oligonucleotideconcentrations of 0 to 2 μM and ACV concentrations from 0 to 16 μM. Asshown in FIG. 11, the effect of the two drugs in combination appeared tobe additive.

Example 18 Activity of G₄-Containing 8-mer Oligonucleotides AgainstHSV-1

A progressive unrandomization strategy [Ecker, D. J. et al., (1993)Nucl. Acids. Res. 21:1853-1956] was used to identify an 8-merphosphorothioate oligonucleotide which was active against HSV-1 in theELISA assay described in Example 3. The “winning” oligonucleotide, ISIS5684, had the sequence GGGGGGTG. The ED50 of this oligonucleotide wasfound to be approximately 0.6 μM.

A series of 8-mer phosphorothioate oligonucleotides containing a G₄sequence were synthesized and tested in the HSV-1 ELISA assay describedin Example 3. These oligonucleotides are shown in Table 14. TABLE 14Anti-HSV Activity of short G₄-containing Oligonucleotides ISIS NO.SEQUENCE 5060 GTGGGGTA 6170 GTGGGGTG 5684 GGGGGGTG 5058 GCGGGGTAAs shown in FIG. 12, all of these oligonucleotides have IC50's below 1μM and are therefore preferred. Several of these 8-mers have anti-HSVactivity greater than that of ISIS 4015, a 20-mer.G₄ Oligonucleotides Active Against HIV:

Example 19 Oligonucleotide Library Synthesis

Phosphorothioate oligonucleotides were synthesized using standardprotocols. Sulfurization was achieved using3H-1,2-benzodithiole-3-one-1,1 dioxide (“Beaucage reagent”) as oxidizingagent. Iyer, R. P., Phillips, L. R., Egan, W., Regan, J. B. & Beaucage,S. L. (1990) J. Org. Chem. 55, 4693-4699. For oligonucleotides withrandomized positions, amidites were mixed in a single vial on the fifthport of the ABI 394 synthesizer. The mixture was tested by coupling todT-CPG, cleaving and deprotecting the product, and analyzing the crudematerial on reversed-phase HPLC. Proportions of the individual amiditeswere adjusted until equal amounts of the four dimers were obtained.DMT-off oligonucleotides were purified by reversed-phase HPLC with agradient of methanol in water to desalt and remove the protectinggroups. Several purified oligonucleotides were analyzed for basecomposition by total digestion with nuclease followed by reversed-phaseHPLC analysis and yielded expected ratios of each base.

Oligonucleotides with the α-configuration of the glycosidic bond weresynthesized as previously described. Morvan, F., Rayner, B., Imbach,J-L., Thenet, S., Bertrand, J-R., Paoletti, J., Malvy, C. & Paoletti, C.(1993) Nucleic Acids Res. 15, 3421-3437. Biotin was incorporated duringchemical synthesis using biotin-linked CPG from Glen Research.Oligonucleotide T₂G₄T₂ (ISIS 5320) was purified by reverse phasechromatography to remove salts and protecting groups and then by sizeexclusion chromatography to purify the tetramer as described in Example21.

Prior to antiviral screening, oligonucleotides were diluted to 1 mMstrand concentration in 40 mM sodium phosphate (pH 7.2), 100 mM KCl andincubated at room temperature overnight. Extinction coefficients weredetermined as described by Puglisi & Tinoco, (1989) In Methods inEnzymology, RNA Processing, eds. Dahlberg, J. E. & Abelson, J. N.(Academic Press, Inc., New York), Vol. 180, pp. 304-324. Samples werefiltered through 0.2 μm cellulose acetate filters to sterilize.

Example 20 Acute HIV-1 Assay

Oligonucleotides were screened in an acute HIV-1 infection assay whichmeasures protection from HIV-induced cytopathic effects. The CEM-SS cellline; Nara, P. L. & Fischinger, P. J. (1988) Nature 332, 469-470; wasmaintained in RPMI 1640 medium supplemented with 10% fetal bovine serum,2 mM glutamine, penicillin (100 units mL⁻¹), and streptomycin (100 μgmL⁻¹). The antiviral assay, using XTT-tetrazolium to quantitatedrug-induced protection from HIV-induced cell killing has beendescribed. White, E. L., Buckheit, Jr., R. W., Ross, L. J., Germany, J.M., Andries, K., Pauwels, R., Janssen, P. A. J., Shannon, W. M. &Chirigos, M. A. (1991) Antiviral Res. 16, 257-266.

Example 21 Characterization of Tetramer

Monomeric and tetrameric forms of oligonucleotides were separated on aPharmacia Superdex HR 10/30 size exclusion column (Pharmacia, Upsalla,Sweden). Running buffer was 25 mM sodium phosphate (pH 7.2), 0.2 mMEDTA. Flow rate was 0.5 mL min⁻¹ and detection was at 260 nm. Monomerand tetramer peaks were integrated and fraction tetramer determined. Forpurification, a Pharmacia Superdex 75 HiLoad 26/60 column was used witha buffer of 10 mM sodium phosphate (pH 7.2) at a flow rate of 2 mLmin⁻¹.

Dissociation of the tetramer was followed after dilution. A 1 mMsolution of oligonucleotide was diluted to 10 μM into PBS (137 mM NaCl;2.7 mM KCl; 1.5 mM potassium phosphate, monobasic; 8 mM sodiumphosphate, dibasic) and incubated at 37° C. Phosphorothioateoligonucleotides having the sequence T₂G₄T₂ in K⁺ and the phosphodiesterT₂G₄T₂ were diluted from solutions in 40 mM sodium phosphate (pH 7.2),100 mM KCl. Oligonucleotide having the sequence T₂G₄T₂ in Na⁺ wasdiluted from a solution in 40 mM sodium phosphate (pH 7.2), 100 mM NaCl.Dissociation as a function of time was followed by size exclusionchromatography.

The tetramer formed was parallel-stranded as determined by analysis ofthe complexes formed by the phosphorothioate oligonucleotides havingT₂G₄T₂ and ^(5′)T₁₃G₄T₄ ^(3′) (SEQ ID NO: 142). Each oligonucleotide waslabeled at the 5′ end with ³²P. Each sample contained 125 μM unlabeledand 15 pM radioactively labeled amounts of one or both of theoligonucleotides. The samples were heated in 50 mM sodium phosphate (pH7.2), 200 mM KCl in a boiling water bath for 15 min then incubated for48 h at 4° C. Samples were analyzed by autoradiography of a 20%non-denaturing polyacrylamide (19:1, acrylamide: bis) gel run at 4° C.in 1×TBE running buffer.

Example 22 Assay of HIV-Induced Cell Fusion

Stochiometric amounts of chronically HIV-1-infected Hut 78 cells(Hut/4-3) and CD4+ HeLa cells harboring an LTR-driven lac z gene wereco-cultured for 20 h in the presence or absence of oligonucleotide.Cells were fixed (1% formaldehyde, 0.2% glutaraldehyde in PBS) andincubated with X-gal until cell-associated color developed. After bufferremoval, a standard o-nitrophenyl-β-D-galactopyranoside was used toquantitate β-galactosidase expression. As a control, HeLa CD4+ cellscontaining the LTR-driven lac Z gene were transfected using the calciumphosphate method with 30 μg of proviral DNA (pNL 4-3). Oligonucleotidewas added immediately after the glycerol shock. Cells were fixed 48 hafter transfection and assayed as described above.

Example 23 Binding of ISIS 5320 to gp120

Direct binding to gp120 was assayed using immobilized gp120 from a CD4capture ELISA kit (American Bio-technologies). Biotinylatedoligonucleotides (biotinylated during synthesis using biotin-linked CPGfrom Glen Research) were incubated in a volume of 100 μL withimmobilized gp120. Following a 1 hour incubation wells were washed and200 μL of streptavidin-alkaline phosphatase (Gibco BRL) diluted 1:1000in PBS added to each well. After a 1 hour incubation at room temperaturewells were washed and PNPP substrate (Pierce) added. Plates wereincubated at 37° C. and absorbance at 405 nm was measured using aTitertek Multiscan MCC/340 ELISA plate reader.

Ability of ISIS 5320 to compete with dextran sulfate for binding togp120 was determined. Biotinylated ISIS 5320 at a concentration of 0.5μM was added to plates containing immobilized gp120 along with dextransulfate at the indicated concentrations (Sigma, M.W. 5000). Following a1 h incubation, the amount of oligonucleotide associated with gp120 wasdetermined as described above.

The site of ISIS 5320 binding to gp120 was determined by competition forbinding of antisera specific for various regions of the protein. Rusche,J. R., et al., (1987) Proc. Natl. Acad. Sci. USA 84, 6924-6928;Matsushita, S., et al., (1988) J. Virol. 62, 2107-2114; Meuller, W. T.,et al., (1986) Science 234, 1392-1395. gp120-coated microtiter plateswere incubated with oligonucleotide at a concentration of 25 μM for 1 hat room temperature. Antisera was added at a dilution of 1:250 and theplates incubated 40 min. The plates were washed four times with PBS andamount of antibody bound quantitated by incubating with proteinA/G-alkaline phosphatase (1:5000, Pierce) in PBS for 1 h at roomtemperature. After one wash with PBS, substrate was added and absorbanceat 405 nm was measured.

Binding of ISIS 5320 to gp120, CD44 and CD4 expressed on cells wasquantitated. HeLa cells harboring an HIV-1 env c gene; Gama Sosa, M. A.,et al., (1989) Biochem. Biophys. Res. Comm. 161, 305-311 and Ruprecht,R. M., et al., (1991) J. Acquir. Immune Defic. Syndr. 4, 48-55; werecultured in DMEM supplemented with 10% FCS and 100 μg μL⁻¹ G-418. Extentof binding to gp120 was detected using 1 μg of FITC-conjugated murineanti-gp120 HIV-1 IIIB mAb IgG (Agmed). CD44 binding was detected using 1μg of FITC-conjugated murine anti-CD44 mAb IgG (Becton-Dickinson). Eachexperiment consisted of 200,000 cells. Cells were washed once in culturemedia with 0.05% NaN₃ then resuspended in 100 μL of media containingoligonucleotide and incubated 15 min at room temperature. Antibody wasadded and the incubation continued for 1 h at 4° C. The cells werewashed twice with PBS and immunofluorescence was measured on aBecton-Dickinson FACScan. Mean fluorescence intensity was determinedusing Lysis software.

CEM-T4 cells; Foley, G. E., et al., (1965) Cancer 18, 522-529; weremaintained in MEM supplemented with 10% FCS. Extent of binding to CD4was determined using 1 μg of Q425, a murine anti-CD4 mAb IgG. Healey,D., et al., (1990) J. Exp. Med. 172, 1233-1242. Cells were harvested andwashed and incubated with oligonucleotide as above. After a 30 minincubation at room temperature with antibody, the cells were washed andincubated with 100 μL of media containing 5 μg of goat F (ab′)₂anti-mouse IgG (Pierce). The cells were incubated 30 min, washed andassociated fluorescence determined as above.

Example 24 Selection and Characterization of T₂G₄T₂

A phosphorothioate oligonucleotide library containing all possiblesequences of eight nucleotides divided into 16 sets, each consisting of4,096 sequences, was prepared as described in Example 19 and screenedfor inhibition of HIV infection as described in Example 21. Results aresummarized in Table 15. TABLE 15 Combinatorial Pools X = A X = G X = C X= T Round 1 NNA NXN NN inactive inactive inactive inactive NNG NXN NNinactive 19.5 (5%) inactive inactive NNC NXN NN inactive inactiveinactive inactive (0%) NNT NXN NN inactive inactive inactive inactive(0%) Round 2 NNG XGN NN 60.7 1.8 (36%)  55.6 56.2 (3%*)  Round 3 NNG GGXNN 8.0 0.5 (94%)   3.1 (19%*) 8.6 Round 4 NAG GGG XN 0.5 0.5 0.5 0.5(87%) NGG GGG XN 0.5 0.6 (99%*) 0.4 0.5 NCG GGG XN 0.7 0.6 0.5 (91%) 0.4NTG GGG XN 0.4 (82%) 0.5 0.4 0.5 Round 5 XTG GGG TN 0.2 (94%) 0.6 (89%*)0.3 (94%) 0.3 (94%) Round 6 TTGGGGTX 0.6 (90%) 0.6 0.5 0.3 (93%)

Random positions, N, are an equimolar mixture of each base. Antiviraldata are reported as the quantity of drug (in μM of oligonucleotidestrand) required to inhibit 50% of virus-induced cell killing (IC₅₀).Error in the IC₅₀ is ±0.1 μM. “Inactive” pools showed no antiviralactivity at 100 μM strand concentration. The % tetramer, determined asdescribed in Example 21, is given in parentheses for selected pools. Anasterisk indicates multiple aggregate species.

The in vitro assay measured protection of cells from HIV-inducedcytopathic effects. White, E. L., et al., (1991) Antiviral Res. 16,257-266. In the initial rounds of selection, antiviral activity wasobserved only in the set containing guanosine in two fixed positions.Subsequent rounds of selection showed that four consecutive Gs providedmaximum antiviral activity. No strong selection preference was observedfor nucleotides flanking the guanosine core. The sequence T₂G₄T₂(oligonucleotide ISIS 5320) was chosen for further study. Theconcentration of ISIS 5320 required for 50% inhibition of virus-inducedcell killing (IC₅₀) was 0.3 μM. The antiviral activity of thisoligonucleotide was not a result of inhibition of cell metabolism;cytotoxic effects were not observed until cells were incubated withapproximately 100 μM ISIS 5320.

Although the oligonucleotide ISIS 5320 has a phosphorothioate backbone,evidence suggests that it adopts a four-stranded, parallel helix as dophosphodiester oligonucleotides of similar sequence. Cheong, C. & Moore,P. B. (1992) Biochemistry 31, 8406-8414; Aboul-ela, F., et al., (1992)Nature 360, 280-282; Sarma, M. H., et al., (1992) J. Biomol. Str. Dyn.9, 1131-1153; and Wang, Y. & Patel, D. J. (1992) Biochemistry 31,8112-8119. The oligonucleotides in the combinatorial library pools thatshow antiviral activity (Table 15) and oligonucleotide ISIS 5320 formmultimeric complexes as shown by size exclusion chromatography (FIG.13). The retention time of the complex was that expected for atetrameric species based on plots of retention time vs. log molecularweight of phosphorothioate oligonucleotide standards (data not shown).The circular dichroism (CD) spectrum of the multimeric form ofoligonucleotide ISIS 5320 is characterized by a peak at 265 nm and atrough at 242 nm (data not shown), similar to the spectra reported byothers for deoxyoligonucleotide tetramers. Sarma, M. H., et al., (1992)J. Biomol. Str. Dyn. 9, 1131-1153; Lu, M., Guo, Q. & Kallenbach, N. R.(1992) Biochemistry 31, 2455-2459; Jin, R., et al., (1992) Proc. Natl.Acad. Sci. USA 89, 8832-8836 and Hardin, C. C., et al., (1992)Biochemistry 31, 833-841. It has been reported that when twophosphodiester oligonucleotides of dissimilar size, but each containingfour or five guanosines in a row, are incubated together, five distinctaggregate species are formed on a non-denaturing gel. Sen, D. & Gilbert,W. (1990) Nature 344, 410-414 and Kim, J., Cheong, C. & Moore, P. B.(1991) Nature 351, 331-332. In principle, only a tetramer of parallelstrands can explain this pattern. When this experiment was performedwith two phosphorothioate oligonucleotides, the antiviraloligonucleotide SIS 5320 and a 21-residue oligonucleotide containing 4guanosines near the 3′ end (^(5′)T₁₃G₄T₄ ^(3′)), the five aggregatespecies expected for a parallel-stranded tetramer were observed on anon-denaturing gel (FIG. 14).

Example 25 The Tetramer is Active Against HIV

Oligonucleotides were screened for antiviral activity as described inExample 22. Samples of ISIS 5320 were diluted from a 1 mM stock solutionthat was at least 98% tetramer. Results showed that the tetramer isstable indefinitely at 1 mM strand concentration; no decrease intetramer was observed over 5 months in a 1 mM sample in buffercontaining 100 mM KCl at room temperature. Upon dilution toconcentrations used in antiviral assays (less than 25 μM) dissociationof the tetramer begins; however, kinetics of the dissociation are veryslow (FIG. 15). Slow kinetics for association and dissociation ofintermolecular G-quartet complexes have been reported. Jin, R., et al.,(1992) Proc. Natl. Acad. Sci USA 89, 8832-8836 and Sen, D. & Gilbert, W.(1990) Nature 344, 410-414. The half life for the dissociation of thepotassium form of ISIS 5320 is about 45 days. During the six-day periodof the acute antiviral assay, at least 70% of the sample remained in thetetramer form whether the sample was prepared in sodium or potassium.Both sodium and potassium forms have the same IC₅₀ values in the acuteantiviral assay, even though potassium preferentially stabilized thetetramer.

Heat denaturation of the tetrameric complex formed by ISIS 5320 beforeaddition to the antiviral assay resulted in loss of activity; antiviralactivity was recovered upon renaturation (data not shown). The strikingdifference in antiviral activity among the initial 16 sets ofoligonucleotides used for combinatorial screening can be explained bythe presence or absence of the G-core and therefore the tetramerstructure (Table 15). In the intial round of screening, approximately12% of the molecules in the active ^(5′)NNGNGNNN^(3′) pool contained atleast four sequential Gs, and size exclusion chromatography showed that5% of the oligonucleotides formed tetramers (Table 15). In contrast, inthe other three round 1 pools where X=G only 0.4% of the moleculescontained at least four sequential Gs and no tetramer was observed. Inother pools, there were no molecules with four consecutive Gs.

Deletion of nucleotides from either end of the ISIS 5320 sequenceresulted in a loss of activity (Table 16). TABLE 16 Sequence IC₅₀ (μM) %tetramer T_(S)T_(S)G_(S)G_(S)G_(S)G_(S)T_(S)T 0.3 98T_(S)T_(S)G_(S)G_(S)G_(S)G_(S)T_(S)T inactive  0 heat denaturedG_(S)G_(S)G_(S)G_(S)T_(S)T 0.5  94* G_(S)G_(S)G_(S)G_(S)T 1.4  61*G_(S)G_(S)G_(S)G 4  29* T_(S)T_(S)G_(S)G_(S)G_(S)G 13  40*T_(S)G_(S)G_(S)G_(S)G inactive  57* T_(S)G_(S)T_(S)G_(S)T_(S)G_(S)T_(S)Ginactive  0 α-T_(S)T_(S)G_(S)G_(S)G_(S)G_(S)T_(S)T 0.5 98α-T_(O)T_(O)G_(O)G_(O)G_(O)G_(O)T_(O)T inactive 97T_(O)T_(O)G_(O)G_(O)G_(O)G_(O)T_(O)T inactive 93T_(S)T_(S)G_(O)G_(O)G_(O)G_(S)T_(S)T 5.0 80T_(O)T_(O)G_(S)G_(S)G_(S)G_(O)T_(O)T inactive 72T_(O)T_(S)G_(O)G_(S)G_(O)G_(S)T_(O)T inactive  9T_(S)T_(O)G_(S)G_(O)G_(S)G_(O)T_(S)T 5.3 83T_(S)T_(S)G_(S)G_(S)G_(S)G_(S)T_(S)T_(S)B 0.4 85

Data from the acute HIV assay for sequence variants and analogs of ISIS5320. Chemical modifications of the oligonucleotide are indicated: “s”phosphorothioate backbone, “o” phosphodiester backbone, “α”,α-configuration of the glycosidic bond; “B” biotin (incorporated duringchemical synthesis using biotin linked CPG from Glen Research).“Inactive” indicates no activity at 25 μM concentration. The % tetramerwas determined as described in Example 21. An asterisk indicates morethan one aggregate species. The phosphorothioate GGGG shows someactivity; two nucleotides on the 3′ side of the four Gs were requiredfor nearly optimal activity. More than one multimeric species wasobserved by size exclusion chromatography for oligonucleotides with theG-core exposed.

The sequence T₂G₄T₂ with a phosphodiester backbone was inactive in theanti-HIV assay, even though the phosphodiester tetramer appears to bekinetically more stable than that formed by the phosphorothioate ISIS5320 (FIG. 15). While not wishing to be bound to a particular theory,two hypotheses are proposed. The phosphorothioate backbone may bemechanistically required or the modified backbone may preventnuclease-mediated degradation of the oligonucleotide.

Oligonucleotide analogs with the glycosidic bond oriented in theα-position are resistant to nuclease degradation. Morvan, F., et al.,(1993) Nucleic Acids Res. 15, 3421-3437. Based on size exclusionchromatography it has been shown that both the phosphorothioateα-oligonucleotide and the phosphodiester α-oligonucleotide formedtetramers however, only the phosphorothioate analog was active againstHIV (Table 16). Assay of oligonucleotides with mixedphosphorothioate-phosphodiester backbones showed that phosphorothioatelinkages at the termini, but not within the G-core, are necessary foractivity. Results are shown in Table 16.

Example 26 Tetramer Inhibits HIV-1 Binding or Fusion to CD4⁺ Cells

The oligonucleotide ISIS 5320 had no effect on chronically infected (H9IIIB) cell models (data not shown) that respond only to inhibitors thatwork at post-integration steps. In a high multiplicity of infection(MOI) experiment performed as described in Srivastava, K. K., et al.,(1991) J. Virol. 65, 3900-3902, ISIS 5320 inhibited production ofintracellular PCR-amplifiable DNA (data not shown), which indicated thatthe compound inhibited an early step of HIV replication, such asbinding, fusion, internalization, or reverse transcription.

The tetramer form of ISIS 5320 also inhibited binding or fusion ofinfectious virus to a CD4⁺ cell. The assay was performed as described inExample 22. HeLa-CD4-LTR-B-gal cells; Kimpton, J. & Emerman, M. (1992)J. Virol. 66, 2232-2239; were incubated for 15 minutes witholigonucleotide at 37° C. prior to the addition of virus. After 1 hour,the cells were washed to remove unbound virus and oligonucleotide.During the incubation period, virus binding and membrane fusion eventsoccur. Srivastava, K. K., et al., (1991) J. Virol. 65, 3900-3902. Extentof infection after 48 hours was determined by quantitation of syncytiaand ELISA as previously described in Kimpton, J. & Emerman, M. (1992) J.Virol. 66, 2232-2239. At a ISIS 5320 concentration of approximately 0.4μM, virus production was reduced to 50% of control (data not shown).Heat-denatured ISIS 5320 and ^(5′)TGTGTGTG^(3′) showed inhibition ofbinding at 5 μM oligonucleotide concentration. These fusion and bindinginhibition experiments strongly suggest that the tetramer form of ISIS5320 inhibits viral infection at a very early step, either duringbinding of the virion to the cell or during the early events of fusionand internalization of the virion.

Example 27 Tetramer Binds to the V3 Domain of gp120

Cellular experiments indicated that ISIS 5320 blocks viral binding orfusion, therefore, the affinities of the ISIS 5320 tetramer for CD4 andgp120 were determined as described in Example 23. Biotinylated ISIS 5320(Table 16) bound to immobilized gp120 with a dissociation constant(K_(d)) of less than 1 μM (FIG. 16). In contrast, a controlphosphorothioate, ⁵T₂A4T2-biotin^(3′), bound weakly to gp120 with anestimated K_(d) of 260 μM. Addition of CD4 at concentrations of up to 50μg mL⁻¹ had no effect on ISIS 5320 binding to gp120 (data not shown).Similar experiments using CD4-coated microtiter plates showed thatbiotinylated ISIS 5320 also associates with CD4; however, the K_(d) ofapproximately 25 μM was considerably weaker than to gp120. The controlbound CD4 only when it was added at very high concentrations (K_(d)approximately 240 μM). In addition, qualitative gel shift assaysperformed as described in Fried, M. & Crothers, D. M. (1981) NucleicAcids Res. 9, 6505-6525, were performed to determine the affinity ofISIS 5320 for other HIV proteins (Tat, p24, reverse transcriptase, vif,protease, gp41), soluble CD4 (sCD4) and non-related proteins (BSA,transferrin and RNase V₁). Both monomeric and tetrameric forms of ISIS5320 bound to BSA and reverse transcriptase. Tetramer-specific bindingwas observed only to gp120 and sCD4.

The V3 loop of gp120 (amino acids 303-338) is considered the principalneutralizing domain of the protein; peptides derived from this regionelicit type-specific neutralizing antibodies that block viral infectionby blocking fusion. (1992) Human Retroviruses and AIDS 1992, eds. Myers,G. et al. (Theoretical Biology and Biophysics, Los Alamos NationalLaboratory, Los Alamos, N. Mex.). The V3 loop of gp120 is also the siteof action of anionic polysaccharides, such as dextran sulfate, thatinhibit viral binding, replication and syncytium formation. Callahan,L., et al., (1991) J. Virol. 65, 1543-1550. Dextran sulfate is acompetitive inhibitor of binding of biotinylated ISIS 5320 to gp120immobilized on a microtiter plate. About 50% of the tetramer binding wasinhibited at a dextran sulfate concentration between 10 and 50 μg mL⁻¹(FIG. 17). Dextran sulfate has been shown to inhibit binding ofgp120-specific antibodies to gp120 in this concentration range.Callahan, L., et al., (1991) J. Virol. 65, 1543-1550.

The oligonucleotide ISIS 5320 also interferes with binding of antiseradirected against the V3 loop region of gp120, but not to antiseraspecific for another region of the protein. Rusche, J. R., et al.,(1987) Proc. Natl. Acad. Sci. USA 84, 6924-6928; Matsushita, S., et al.,(1988) J. Virol. 62, 2107-2114 and Meuller, W. T., et al., (1986)Science 234, 1392-1395. The control oligonucleotide had no effect onantibody binding.

The tetramer also binds to the V3 loop of gp120 expressed on cells.Binding of a monoclonal antibody specific for the V3 loop of gp120 wasinhibited by ISIS 5320 at a concentration of approximately 0.5 μM(K_(i)) determined using immunofluorescent flow cytometry (FIG. 18). Thecontrol oligonucleotide had little effect on binding at concentrationsup to 50 μM. Neither oligonucleotide significantly decreased binding ofantibodies directed to human CD44 on the same cells or to CD4; Healey,D., et al., (1990) J. Exp. Med. 172, 1233-1242. on CEM-T4 cells.

Phosphorothioate oligonucleotides of at least 15 nucleotides are knownto be non-sequence-specific inhibitors of HIV. Stein, C. A., et al.,(1991) J. Acquir. Immune Defic. Syndr. 4, 686-693. In the acute assaysystem used here, previously tested phosphorothioate oligonucleotides of18 to 28 nucleotides in length have IC₅₀ values between 0.2 and 4 μM.Vickers, T., et al., (1991) Nucleic Acids Res. 19, 3359-3368. Stein andco-workers have shown that phosphorothioate oligonucleotides of at least18 nucleotides in length, bind to the V3 loop of gp120 (40), and to theCD4 receptor and other cell surface antigens. Stein, C. A., et al.,(1991) J. Acquir. Immune Defic. Syndr. 4, 686-693. Variation in thebinding and antiviral activities of long mixed seqence oligonucleotideslikely result from folding into unknown structures with varyingaffinities for membrane surface proteins. In contrast, ISIS 5320 adoptsa defined tetrameric structure. The antiviral activity is 2- to 25-foldbetter, on a weight basis, than that of longer linear oligonucleotides.

ELISA assays were performed to determine whether ISIS 5320 was capableof blocking the interaction between CD4 and gp120 (data not shown).Addition of increasing amounts of ISIS 5320 decreased binding of CD4 toimmobilized gp120; 50% of binding was inhibited at a concentration ofapproximately 2.5 μM. The control oligonucleotide (^(5′)TGTGTGTG^(3′))had no effect on the CD4/gp120 interaction. These results were confirmedin a gp120-capture ELISA assay in which the microtiter plates werecoated with CD4 (IC₅₀ approximately 20 μM). Compounds that bind to theV3 loop of gp120 can inhibit fusion without completely blocking theinteraction between CD4 and gp120. Callahan, L., et al., (1991) J.Virol. 65, 1543-1550. Unlike ISIS 5320, dextran sulfate does not preventthe gp120/CD4 interaction in an ELISA assay even at concentrations10,000-fold above its IC₅₀ Callahan, L., et al., (1991) J. Virol. 65,1543-1550.

The tetrameric form of phosphorothioate T₂G₄T₂ blocks cell-to-cell andvirion-to-cell spread of HIV infection by binding to the gp120 V3 loop.The tetramer provides a rigid, compact structure with a highthio-anionic charge density that may be the basis for its stronginteraction with the cationic V3 loop. Although the V3 loop is ahypervariable region, the functional requirement for cationic residuesin the V3 loop may limit the virus's capability to become resistant todense poly-anionic inhibitors. Compounds derived from the G-quartetstructural motif are potential candidates for use in anti-HIVchemotherapy.

1. A chemically modified oligonucleotide having no more than about 27nucleic acid base units, said oligonucleotide comprising at least oneGGGG sequence or at least two GGG sequences and a sufficient number offlanking nucleotides to significantly inhibit the activity of a virus orphospholipase A₂ or to modulate the telomere length of a chromosome. 2.An oligonucleotide of claim 1 wherein significant inhibition of viral orenzyme activity is at least 50% inhibition.
 3. An oligonucleotide ofclaim 1 wherein the virus is HIV, HSV, HCMV or influenza virus.
 4. Anoligonucleotide of claim 3 wherein the virus is HSV.
 5. Anoligonucleotide of claim 4 wherein the oligonucleotide is selected fromthe group consisting of: SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 8, SEQID NO: 12, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 47, SEQ IDNO: 48 and SEQ ID NO:
 50. 6. An oligonucleotide of claim 4 having asequence shown in Table
 8. 7. An oligonucleotide of claim 6 having asequence selected from the group consisting of SEQ ID NO: 124, SEQ IDNO:126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130and SEQ ID NO:
 133. 8. An oligonucleotide of claim 1 having the sequence(N_(X)G₄N_(Y))_(Q) wherein X and Y are independently 1 to 8 and Q is 1to
 4. 9. An oligonucleotide of claim 8 having the sequence NNGGGGNN. 10.An oligonucleotide of claim 9 which has at least one phosphorothioateintersugar linkage and which has the sequence GNGGGGTN.
 11. Anoligonucleotide of claim 1 having the sequence (G₄N_(X)G₄)_(Q) wherein Xis 1 to 8 and Q is 1 to
 3. 12. An oligonucleotide of claim 1 having thesequence (N_(X)G₃₋₄)_(Q)N_(X) wherein X is 1 to 8 and Q is 1 to
 6. 13.An oligonucleotide of claim 1 which has at least one phosphorothioateintersugar (backbone) linkage.
 14. An oligonucleotide of claim 1 whereineach of the nucleosides is in the alpha (α) anomeric configuration. 15.An oligonucleotide of claim 1 which is a chimeric oligonucleotide.
 16. Aphosphorothioate oligonucleotide having SEQ ID NO:
 21. 17. Aphosphorothioate oligonucleotide having the sequence TTGGGGTT.
 18. Theoligonucleotide of claim 17 wherein each of the nucleotides of theoligonucleotide is in the alpha (α) anomeric configuration.
 19. A methodfor inhibiting the activity of a virus comprising contacting the viruswith a chemically modified oligonucleotide having no more than 27nucleic acid base units comprising at least one GGGG sequence and atleast two GGG sequences and a sufficient number of flanking nucleotidesto significantly inhibit the activity of the virus.
 20. The method ofclaim 19 wherein significant inhibition of viral activity is at least50% inhibition.
 21. The method of claim 19 wherein the virus is HIV,HSV, HCMV or influenza virus.
 22. The method of claim 21 wherein thevirus is HSV.
 23. The method of claim 22 wherein the oligonucleotide isselected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 1, SEQID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ IDNO: 47, SEQ ID NO: 48 and SEQ ID NO:
 50. 24. The method of claim 22wherein the oligonucleotide has a sequence shown in Table
 8. 25. Themethod of claim 24 wherein the oligonucleotide has a sequence selectedfrom the group consisting of SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 133.26. The method of claim 19 wherein said oligonucleotide has the sequence(N_(X)G₄N_(Y))_(Q) wherein X and Y are independently 1 to 8 and Q is 1to
 4. 27. The method of claim 26 wherein said oligonucleotide has thesequence NNGGGGNN.
 28. The method of claim 27 wherein theoligonucleotide has at least one phosphorothioate intersugar linkage andthe sequence GNGGGGTN.
 29. The method of claim 19 wherein saidoligonucleotide has the sequence (G₄N_(X)G₄)_(Q) wherein X is 1 to 8 andQ is 1 to
 3. 30. The method of claim 19 wherein said oligonucleotide hasthe sequence (N_(X)G₃₋₄)_(Q)N_(X) wherein X is 1 to 8 and Q is 1 to 6.31. The method of claim 19 wherein said oligonucleotide comprises asequence identified in Table 1, Table 2 or Table
 3. 32. The method ofclaim 19 wherein said oligonucleotide has at least one phosphorothioateintersugar (backbone) linkage.
 33. The method of claim 19 wherein eachof the nucleosides of the oligonucleotide is in the alpha (α) anomericconfiguration.
 34. The method of claim 19 wherein the oligonucleotide isa chimeric oligonucleotide.
 35. A method for inhibiting the activity ofa virus comprising contacting the virus with a phosphorothioateoligonucleotide having SEQ ID NO:
 21. 36. A method for inhibiting theactivity of a virus comprising contacting the virus with aphosphorothioate oligonucleotide having the sequence TTGGGGTT.
 37. Themethod of claim 36 wherein each of the nucleotides of theoligonucleotide is in the alpha (α) anomeric configuration.
 38. Themethod of claim 36 wherein the virus is HIV.
 39. A method for inhibitingphospholipase A₂ enzyme activity comprising contacting a cell with achemically modified oligonucleotide having no more than about 27 nucleicacid base units comprising at least one GGGG sequence or at least twoGGG sequences and a sufficient number of flanking nucleotides tosignificantly inhibit the activity of phospholipase A₂.
 40. The methodof claim 39 wherein the phospholipase A₂ enzyme activity is inhibited bygreater than 50%.
 41. The method of claim 39 wherein saidoligonucleotide comprises a sequence selected from the group consistingof: SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26, SEQ IDNO:42, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:68, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:83, SEQ ID NO:97, SEQ IDNO:98, SEQ ID NO:99, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ IDNO:107 and TGGGG.
 42. The method of claim 39 wherein saidoligonucleotide has at least one phosphorothioate intersugar (backbone)linkage.
 43. A method of treating a viral-associated disease comprisingadministering to an animal having a viral-associated disease atherapeutically effective amount of a chemically modifiedoligonucleotide having no more than about 27 nucleic acid base unitscomprising at least one GGGG sequence and at least two GGG sequences anda sufficient number of flanking nucleotides to significantly inhibit theactivity of the virus.
 44. The method of claim 43 wherein significantinhibition of viral activity is at least 50% inhibition.
 45. The methodof claim 43 wherein the virus is HIV, HSV, HCMV or influenza virus. 46.The method of claim 45 wherein the virus is HSV.
 47. The method of claim46 wherein the oligonucleotide is selected from the group consisting of:SEQ ID NO: 21, SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16,SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 50.48. The method of claim 46 wherein the nucleotide has a sequence shownin Table
 8. 49. The method of claim 48 wherein the oligonucleotide has asequence selected from the group consisting of SEQ ID NO: 124, SEQ IDNO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130and SEQ ID NO:
 133. 50. The method of claim 43 wherein saidoligonucleotide has the sequence (N_(X)G₄N_(Y))_(Q) wherein X and Y areindependently 1 to 8 and Q is 1 to
 4. 51. The method of claim 50 whereinsaid oligonucleotide has the sequence NNGGGGNN.
 52. The method of claim51 wherein said oligonucleotide has at least one phosphorothioateintersugar linkage and the sequence GNGGGGTN.
 53. The method of claim 43wherein said oligonucleotide has the sequence (G₄N_(X)G₄)_(Q) wherein Xis 1 to 8 and Q is 1 to
 3. 54. The method of claim 43 wherein saidoligonucleotide has the sequence (N_(X)G₃₋₄)_(Q)N_(X) wherein X is 1 to8 and Q is 1 to
 6. 55. The method of claim 43 wherein saidoligonucleotide comprises a sequence identified in Table 1, Table 2 orTable
 3. 56. The method of claim 43 wherein said oligonucleotide has atleast one phosphorothioate intersugar (backbone) linkage.
 57. The methodof claim 43 wherein each of the nucleotides of the oligonucleotide is inthe alpha (α) anomeric configuration.
 58. The method of claim 43 whereinthe oligonucleotide is a chimeric oligonucleotide.
 59. A method oftreating a viral-associated disease comprising contacting the virus witha phosphorothioate oligonucleotide having SEQ ID NO:
 21. 60. A method oftreating a viral-associated disease comprising contacting the virus witha phosphorothioate oligonucleotide having the sequence TTGGGGTT.
 61. Themethod of claim 60 wherein each of the nucleotides of theoligonucleotide is in the alpha (α) anomeric configuration.
 62. Themethod of claim 60 wherein the virus is HIV.
 63. A method of treating aninflammatory disease or a neurological disorder associated withphospholipase A₂ enzyme activity comprising administering to an animalhaving such an inflammatory disease or neurological disease atherapeutically effective amount of a chemically modifiedoligonucleotide having no more than about 27 nucleic acid base unitscomprising at least one GGGG sequence and at least two GGG sequences anda sufficient number of flanking nucleotides to significantly inhibit theactivity of phospholipase A₂.
 64. The method of claim 63 whereinsignificant inhibition of enzyme activity is at least 50% inhibition.65. The method of claim 63 wherein said oligonucleotide comprises asequence selected from the group consisting of: SEQ ID NO:12, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:42, SEQ ID NO:47, SEQ IDNO:50, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:83, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO: 104, SEQ ID NO: 105, SEQ ID NO:106, SEQ ID NO:107 and TGGGG.
 66. Amethod of modulating telomere length of a chromosome comprisingcontacting a chromosome with a chemically modified oligonucleotide 6 to27 nucleic acid base units in length having the sequence(N_(X)G₃₋₄)_(Q)N_(X) wherein X is 1-8 and Q is 1-5.
 67. A method forinhibiting the division of a malignant cell comprising contacting amalignant cell with a chemically modified oligonucleotide having 6 to 27nucleic acid base units and having the sequence (N_(X)G₃₋₄)_(Q)N_(X)wherein X is 1-8 and Q is 1-5.
 68. A compound comprising a G-quartetstructure of phosphorothioate oligonucleotides each oligonucleotidehaving the sequence TxG4Ty where x and y are independently 0 to
 8. 69.The compound of claim 68 wherein the nucleotides of at least one of theoligonucleotides of the G-quartet structure are in the alpha (α)anomeric configuration.
 70. The compound of claim 68 wherein x is 2 andy is
 2. 71. The compound of claim 68 wherein x is 0 and y is
 2. 72. Thecompound of claim 68 wherein x is 3 and y is
 3. 73. The compound ofclaim 68 wherein each oligonucleotide has the sequence (TxG4Ty)q where xand y are independently 0 to 8 and q is from 1 to
 10. 74. A method forinhibiting the activity of human immunodeficiency virus comprisingadministering to a cell infected with said virus a compound comprising aG-quartet structure of phosphorothioate oligonucleotides eacholigonucleotide having the sequence TxG4Ty where x and y areindependently 0 to 8 in an amount sufficient to inhibit the activity ofthe virus.
 75. The method of claim 74 wherein inhibition of viralactivity is at least 50% inhibition.
 76. The method of claim 74 whereina compound in which x is 2 and y is 2 is administered to a cell infectedwith human immunodeficiency virus.
 77. The method of claim 75 wherein acompound in which x is 0 and y is 2 is administered to a cell infectedwith human immunodeficiency virus.
 78. The method of claim 75 wherein acompound in which x is 3 and y is 3 is administered to a cell infectedwith human immunodeficiency virus.
 79. A method for treating a patientinfected with human immunodeficiency virus comprising administering tosaid patient a compound comprising a G-quartet structure ofphosphorothioate oligonucleotides having the sequence TxG4Ty where x andy are independently 0 to 8 in an amount sufficient to inhibit theactivity of the virus.
 80. The method of claim 79 wherein a compound inwhich x is 2 and y is 2 is administered to said patient infected withhuman immunodeficiency virus.
 81. The method of claim 79 wherein acompound in which x is 0 and y is 2 is administered to said patientinfected with human immunodeficiency virus.
 82. The method of claim 79wherein a compound in which x is 3 and y is 3 is administered to saidpatient infected with human immunodeficiency virus.
 83. A pharmaceuticalcomposition comprising a compound comprising a G-quartet structure ofphosphorothioate oligonucleotides having the sequence TxG4Ty where x andy are independently 0 to 8 and a pharmaceutically acceptable carrier.84. A prophylactic device coated with a compound comprising a G-quartetstructure of phosphorothioate oligonucleotides having the sequenceTxG4Ty where x and y are independently 0 to 8.